Solid Phase Extraction, Derivatization with Crown Ethers, and Mass Spectrometry, Methods, Reagents and Kits

ABSTRACT

The present disclosure is directed to methods reagents and kits for solid phase extraction, derivatization with crown ether containing derivatizing agents, and mass spectrometry of the derivatized analytes.

PRIORITY

This patent application claims the right of priority pursuant to 35U.S.C. §119(e) and is entitled to the benefit of the filing date of U.S.Provisional Patent Application Ser. No. 62/139,692, filed on Mar. 28,2015, the content of which is hereby expressly incorporated by referencein its entirety.

INTRODUCTION

There is a need to obtain fast, streamlined, and automated methods fordetection of particular analytes. In the medical context, such methodswould facilitate the detection of analytes, such as drugs, hormones,signaling agents, and amino acids. In the agricultural and public healthcontext, the detection of residual levels of antibiotics, pesticides orother contaminants is integral to the safety of the food we eat andwater we drink. Furthermore, recent changes in the standards for foodlabeling, such as “hormone free” or “organic” have created a need forstreamlined testing in the agricultural realm.

In many cases clinical quantification of particular analytes was carriedout via radio immunoassays (RIA). In recent years, due to the technologyadvancement in Mass Spectroscopy, there is a gradual shift in testingplatform from RIA to liquid chromatography mass spectrometry (LCMS).Often times, the LCMS based assays provide a fast, sensitive, andanalyte specific readout, which an RIA assay may lack. However, certainanalytes may be difficult to detect for a multitude of reasons. Forinstance, some analytes are difficult to detect as they are prone to beoxidized and degraded. Further, the current art in quantification ofcertain analytes often involves complex and cumbersome extractionprocedures with unstable extraction recovery. For instance, unstableextraction recovery gives rise to results which are often unreliable,and with high lower limits of quantification (LLOQ); for example an LLOQin >10 ng/mL range. Also, the existing extraction process is unable toprovide a reliable sample for derivatization.

Drugs include both illegal and legal drugs and metabolites orderivatives thereof. The detection of these compounds for forensic orprescription compliance are very important. Again, these drugs may notbe stable in bodily fluids and therefore, detection may be difficult. Inany event, there is a great need for a fully automated and simplifieddetection/quantification procedure which minimizes the room for humaninvolvement or error.

SUMMARY

This invention document provides materials and methods that can be usedto measure the levels of mono-acylatable, mono-amine-containing, ormono-phenol-containing analytes. For instance, analytes of interestinclude neurotransmitters, hormones, estrogen hormones (estrone,estradiol, estriol), cannabinoids such as Δ-9-tetrahydrocannabinol (THC)and HU210, serotonin, amino acids, BPA and many other primary aminecontaining, or phenol containing molecules in a biological sample.

This invention document also provides materials and methods that can beused to measure cis-diene containing analytes, such as vitamin-D, itsanalogs and metabolites (e.g., 25-OH D₃, 25-OH D₂, 24,25-(OH)₂ D₃,1,25-(OH)₂ D₃, 1,25-(OH)₂ D₂).

This invention document also provides materials and methods that can beused to derivatize and measure ketone, or aldehyde containing analytes,such as testosterone, progesterone, corticosterone, and many of thesteroid hormones.

The desired analyte can be selectively and sensitively detected andmeasured by mass spectrometry, including tandem mass spectrometrytechnologies. The entire quantification process includes a samplepreparation process that employs a solid phase extraction to capture theanalyte, followed by a chemical derivatization of the analyte, thenquantification via LC-MS/MS technologies, as described herein.

It was discovered during this work that the combination of solid phaseextraction with a chemical derivatization with a crown ether containingreagent and LC-MS/MS provides analyte specific, sensitive, accurate,stable and robust measurements of levels of analytes at pg/ml, fg/ml, orlower in blood plasma/serum sample and in the fg/ml range for detectionof analytes from oral fluid. Quantification methods for were alsodeveloped with similar sensitivity and accuracy.

Sensitive and accurate measurements of these analytes at levels relevantto the clinical setting is useful, particularly at femto-gram or lowpico-gram per milliliter range, at small sample size (100 uL or less ofblood plasma/serum sample), with a simple process which can be easilyconfigured to be automated, with an analyte specific results, withoutany analyte crossovers that are often seen under other analyticaltechniques such as radioimmunoassay (RIA).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, demonstrates a specific embodiment of a derivatizing agent(100), having a crown-ether component (i.e., a sensitivity booster)(101), bound to a linker (i.e., the connector) (102), which will in turnbind the analyte (the derivatization functional group) (103).

FIGS. 2A and B demonstrate the linear regression of THC and HU210 spikedsamples tested according to the present methods. FIG. 2A provides thelinear regression of THC and FIG. 2B provides the linear regression ofHU210.

FIGS. 3A and B demonstrate the linear regression of estrone andestradiol samples tested according to the present methods, and theirchromatograms. FIG. 3A provides the linear regression and FIG. 3Bprovides the chromatograms.

FIGS. 4A and B demonstrate chromatograms of estrone and estradiolsamples tested according to the present methods, and the amounts ofestradiol/estrone in samples taken from 10 individuals. FIG. 4A providesthe chromatogram and FIG. 4B provides a graph of the levels in eachsample.

DETAILED DESCRIPTION

The present methods are directed to methods of extraction,derivatization, and detection and/or quantification of analytes from asample. The present methods employ a sample clean-up process via solidphase extraction (SPE), chemical derivatization process using crownethers in conjunction with detection or quantification of analytes ofinterest to obtain analyte specific, robust, fast, sensitive, andaccurate results. The derivatization of the analyte of interest shiftsthe analyte out of the region where it might potentially overlap withother biological agents found in the sample, enhances sensitivity of theanalyte by trapping easily detected cations, and makes lipophilicanalytes more hydrophilic and thus able to be analyzed. Thus, duringdetection, other biological molecules or contaminants which wouldordinarily interfere with the accurate quantification of the analyte ofinterest are detected at a significantly different range of the spectrum(or eluted completely), allowing the accurate quantification of theanalyte of interest. Furthermore, detection of the derivatized analyteis sensitized by detection of the trapped cations. Finally,hard-to-resolve lipophilic structures may be analyzed by this techniquedue to the unique hydrophobic/hydrophilic structure of the crown ethers.Such analyte detection is useful, e.g., in a clinical, a veterinarian, aforensic, and/or an environmental setting.

The present methods may include derivatization of the analytes ofinterest, and determination of the amount of one or more of the analytesby at least one of chromatography and/or mass spectrometry. In oneembodiment, the present methods may also include a solid phaseextraction (SPE) of the analyte from a biological sample. In one aspect,it is recommended that cation exchange based SPE may be used for captureamine containing analytes. For non-amine containing analytes, a reversephase based SPE may be employed with a protein crash with or without aphospholipid removal process, where such sorbent may be C4-C18 alkylbounded silica, phenyl bounded silica, biphenyl bonded silica, or otherpolymer sorbent. In other embodiments the final elution buffer with highpH (8≦pH≦14) may be used to elute amine containing analyte(s) from thecation exchange solid phase and optionally in situ derivatization of theanalyte(s) of interest, cleanly preparing the derivative of the analytefor detection and/or quantification.

In additional embodiments, the non-amine containing analytes may beelute off from the SPE sorbent via a non-alcoholic, water miscible,organic solvent, such as acetonitrile, DMF, acetone, 1,4-dioxane, THF,NMP, DMSO, and etc., followed by direct derivatization of the analyteunder a pH buffered condition. In a preferred embodiment, the presentmethods for detection of an amine containing analyte include cationexchange SPE, in situ derivatization with a crown ether and thenquantification via LC-MS of the analyte of interest; permitting inlinetransitioning from biological sample to initial analyte extraction todetection and/or quantification. Similarly, for the preferred embodimentfor detection of a non-amine containing analyte, present methods includereverse phase based SPE, organic elution followed by directderivatization with a crown ether, then completed by LC-MS/MSquantification. Or in another embodiment, an anion exchange based SPEmay be used for detection of an analyte with a carboxylic functiongroup, with a high pH elution buffer mixed with suitable organicsolvents such as acetonitrile, DMF, acetone, 1,4-dioxane, THF, NMP,DMSO, and etc., followed by direct derivatization of the analyte.

Also presented are kits for a detection and/or quantification assay.Such kits may include SPE columns (cartridges) and a crown etherderivatization reagent. Optionally, such kits may also include a SPEconditioning solvent, loading buffer, washing buffer, and an eluentbuffer, separately or together, and an HPLC column.

The present methods may detect the presence of a wide array of analytes.An analyte is any compound or composition of interest to be found in thesample of interest. More specifically, an analyte of interest is acompound having primary and or secondary amine groups, or that ismono-acylatable, a mono-amine compound, and or, a phenolic moiety. It ispreferred that there is a single acylation or amine. An analyte ofinterest can have a hydrazine, hydrazide, or hydroxylamine group. Ananalyte as disclosed herein may be a drug (illegal and FDA approved) anda derivative or a metabolite thereof, a pesticide and a derivative ormetabolite thereof, an environmental contaminate and a derivative ormetabolite thereof, or a biologic compound such as, e.g., a hormone, acytokine, a signaling agent, an amino acid, cholesterol or one of itsderivatives, a fatty acid or a glycolipid, and a derivative ormetabolite thereof.

In one embodiment, an analyte is vitamin D, its analogs and metabolites(for example, some members described herein without limitation include:25-OH vitamin D₃, 25-OH D₂, 24,25-(OH)₂ vitamin D₃, 1,25-(OH)₂ vitaminD₃, and 1,25-(OH)₂ vitamin D₂, Cholecalciferol,25-Hydroxycholecalciferol, 1α,25-Dihydroxycholecalciferol,Ergocalciferol, 1α,25-Dihydroxyergocalciferol,22,23-Dihydroergocalciferol, 1α,24R,25-Trihydroxycholecalciferol,(6Z)-tacalciol, Tachysterol₃, Isovitamin D₃, Dihydrotachysterol₃,calcitrol, calcipotriol, etc.).

In one embodiment, an analyte is a monoamine neurotransmitter, or one ofits derivatives or metabolites.

In another embodiment, an analyte is an estrogen including Estrone (E1),Estradiol (E2), Estriol (E3), and Estetrol (E4).

In another embodiment, an analyte is a phytoestrogen, includingdaidzein, formononetin, genistein, biochanin A, coumestrol,4′-methoxycoumestrol, repensol, trifoliol, or 17-beta-estradiol.

In another embodiment, an analyte is a cannabinoid or one of itsderivatives or metabolites. Examples of cannabinoids or one of itsderivatives or metabolites include a Cannabigerol-type (CBG) cannabinoidsuch as, e.g., Cannabigerol, Cannabigerol monomethyl ether,Cannabinerolic acid A, Cannabigerovarin, Cannabigerolic acid A,Cannabigerolic acid A monomethyl ether, and Cannabigerovarinic acid A; aCannabichromene-type (CBC) cannabinoid, such as, e.g.,(±)-Cannabichromene, (±)-Cannabichromenic acid A,(±)-Cannabivarichromene, (±)-Cannabichromevarin, or(±)-Cannabichromevarinic acid A; a Cannabidiol-type (CBD) cannabinoidsuch as, e.g., (−)-Cannabidiol, Cannabidiol momomethyl ether,Cannabidiol-C4, (−)-Cannabidivarin, Cannabidiorcol, Cannabidiolic acid,Cannabidivarinic acid; a Cannabinodiol-type (CBND) cannabinoid such as,e.g., Cannabinodiol or Cannabinodivarin; a Tetrahydrocannabinol-type(THC) cannabinoid such as, e.g., Δ9-Tetrahydrocannabinol,Δ9-Tetrahydrocannabinol-C4, Δ9-Tetrahydrocannabivarin,Δ9-Tetrahydrocannabiorcol, Δ9-Tetrahydro-cannabinolic acid A,Δ9-Tetrahydro-cannabinolic acid B, Δ9-Tetrahydro-cannabinolic acid-C4 A,Δ9-Tetrahydro-cannabinolic acid-C4 B, Δ9-Tetrahydro-cannabivarinic acidA, Δ9-Tetrahydro-cannabiorcolic acid A, Δ9-Tetrahydro-cannabiorcolicacid B, (−)-Δ8-trans-(6aR,10aR)-Δ8-Tetrahydrocannabinol,(−)-Δ8-trans-(6aR,10aR)-Tetrahydrocannabinolic acid A,(−)-(6aS,10aR)-Δ9-Tetrahydrocannabinol; a Cannabinol-type (CBN)cannabinoid such as, e.g., Cannabinol, Cannabinol-C4, Cannabivarin,Cannabinol-C2, Cannabiorcol, Cannabinolic acid A, Cannabinol methylether; a Cannabitriol-type (CBT) cannabinoid such as, e.g.,(−)-(9R,10R)-trans-Cannabitriol, (+)-(9S,10S)-Cannabitriol,(±)-(9R,10S/9S,10R)-Cannabitriol,(−)-(9R,10R)-trans-10-O-Ethyl-cannabitriol,(±)-(9R,10R/9S,10S)-Cannabitriol-C3,8,9-Dihydroxy-Δ6a(10a)-tetrahydrocannabinol, Cannabidiolic acid Acannabitriol ester,(−)-(6aR,9S,10S,10aR)-9,10-Dihydroxy-hexahydrocannabinol,(−)-6a,7,10a-Trihydroxy-Δ9-tetrahydrocannabinol,10-Oxo-Δ6a(10a)-tetrahydrocannabinol; a Cannabielsoin-type (CBE)cannabinoid such as, e.g., (5aS,6S,9R,9aR)-Cannabielsoin,(5aS,6S,9R,9aR)-C3-Cannabielsoin, (5aS,6S,9R,9aR)-Cannabielsoic acid A,(5aS,6S,9R,9aR)-Cannabielsoic acid B, (5aS,6S,9R,9aR)-C3-Cannabielsoicacid B, Cannabiglendol-C3, Dehydrocannabifuran, or Cannabifuran; anIsocannabinoid such as, e.g.,(−)-Δ7-trans-(1R,3R,6R)-Isotetrahydrocannabinol,(±)-Δ7-1,2-cis-(1R,3R,6S/1S,3S,6R)-Isotetrahydrocannabivarin, or(−)-Δ7-trans-(1R,3R,6R)-Isotetrahydrocannabivarin; a Cannabicyclol-type(CBL) cannabinoid such as, e.g., (±)-(1 aS,3aR,8bR,8cR)-Cannabicyclol,(±)-(1aS,3aR,8bR,8cR)-Cannabicyclolic acid A, or(±)-(1aS,3aR,8bR,8cR)-Cannabicyclovarin; a Cannabicitran-type (CBT)cannabinoid such as, e.g., Cannabicitran; and a Cannabichromanone-type(CBCN) cannabinoid such as, e.g., Cannabichromanone,Cannabichromanone-C3, or Cannabicoumaronone. Furthermore, the analytemay be a synthetic cannabinoid such as HU210, cannabicyclohexanol,JWH-073, JWH-018, AM-2201, CP-47,497 (as well as its derivatives andmetabolites), JWH-015, JWH-081, JWH-133, JWH-200, JWH-250, JWH-398,JTE-907, CP 55,244, CP 55,940, HU210, HU-211, WIN 55,212-2, AM-694,AM-1248, AM-2201, AM-2233, EAM-2201, MAM-2201, MN-25, NNE1, 2NE1,UR-144, 5F-UR-144, XLR11, AKB48, AKB-NI, BAY 38-7271, BB-22, CB-25,CB-52, AB-001, AB-034, PB-22, 5F-PB-22, RCS-4, STS-135, URB-597, andURB-754.

In another embodiment, an analyte is a thyroid hormone or one of itsderivatives or metabolites. Examples of thyroid hormones or one of itsderivatives or metabolites include 3,3′,5-triiodothyronine (T₃),3,5,5′-triiodothyronine (rT₃), and thyroxine (T₄).

In another embodiment, an analyte is an opiate, opioid or one of itsderivatives or metabolites. Examples, of opiates or opioids or one ofits derivatives or metabolites include the naturally-occurringbenzylisoquinoline alkaloids (morphine, and oripavine), thesemi-synthetic derivatives (hydromorphone, and oxymorphone), and thesynthetic opioids (e.g., buprenorphine, etorphine, pentazocine).

In another embodiment, an analyte is arylcyclohexylamine or one of itsderivatives or metabolites. Examples of an arylcyclohexylamine includeTiletamine, 3-Methoxetamine (MXE), Methoxyketamine, N-ethylnorletamine(Ethketamine)

In another embodiment, an analyte is an Amphetamine. Examples ofAmphetamines are Amphetamine (itself), methamphetamine, ephedrine,cathinone, 3,4-methylenedioxy-N-methylamphetamine (MDMA, “Ecstasy”), and2,5-Dimethoxy-4-methylamphetamine (DOM, or “STP”).

In another embodiment, an analyte is an amino acid, an artificial aminoacid, or a small peptide. Examples of the amino acid include but are notlimited to: glycine, alanine, phenylalanine, tyrosine, GABA, tryptophan,cysteine, serine, valine, leucine, isoleucine, lysine, methionine,histidine, arginine, aspartic acid, asparagine, glutamic acid,glutamine, proline, and threonine.

Sample Preparation

Aspects of the present specification disclose, in part, a test sample. Atest sample refers to any sample that may contain an analyte ofinterest. A test sample may be a biological sample, that is, a sampleobtained from any biological source, such as an animal, a plant, afungus, a microorganism, a cell culture, an organ culture, etc. Inaspects of this embodiment, a biological sample includes a blood sampleincluding a whole blood sample, a dry blood sample, a plasma sample, ora serum sample, a saliva sample, a lachrymal sample, a semen sample, aurine sample, cerebrospinal fluid sample, a bile sample, an embryonicfluid sample, a tissue sample, or any other sample that can be obtained,extracted or isolated from a biological source. Such biological samplesmay be obtained, for example in a medical or clinical setting, from apatient; that is, a living person, male or female, presenting oneself ina clinical setting for diagnosis, prognosis, or treatment of a diseaseor condition. The sample is preferably obtained from a patient, forexample, a plasma specimen. The plasma specimen may be taken with orwithout the use of anticoagulants.

Such biological samples may be obtained, for example in a veterinariansetting, from an animal; that is, a pet animal, or a farm animal orlivestock, a fish, or any other creatures that live in fresh water,ocean or sea, male or female, presenting oneself in a veterinariansetting for diagnosis, prognosis, prevention, or treatment of a diseaseor condition. The sample is preferably obtained from an animal, forexample, a plasma specimen. The plasma specimen may be taken with orwithout the use of anticoagulants.

A test sample may be obtained from a plant or any vegetation source, inagricultural or environmental setting, from a leave, or a flower, or astem, or a fruit, or a seed, or sprout, or a bark, or a root, etc.

A test sample may be obtained from a dead human body, or a dead animal,or a dead plant, or remains of a once living body, as in a forensicsetting, or in an agricultural setting, or in an environmental setting,or in an archeological setting. A testing sample may be a blood sample,or a dry blood sample, or any other body fluid sample (e.g., saliva orother oral fluid), or any other dry body fluid sample, or a body tissuesample taken from anywhere of the body or remains of a dead human,animal, or plant.

A test sample may be an environmental sample. Environmental samples aresamples taken from dirt, plant matter, or fluid sources (such as groundwater, oceans, or rivers etc.). Dirt (aka “soil samples”) may be takenfrom agricultural sites or sites of environmental interest and may havethe analyte extracted, including the removal of particulate matter.

Samples may be obtained by any known means. The sample may be preservedor pre-treated to ensure stability of the analyte of interest. Suchpreservation may be accomplished by chemical (such as hydrolysis or pHadjustment) or physical processes (such as refrigeration or freezing).When a sample is a solid or a tissue, it can be grounded, or extracted,or purified, or filtered, or centrifuged, to isolate the analyte ofinterest from the interfering components. Or a sample is a liquid,preferably, it is dissolved, or suspended, in a solution (or “loadingbuffer”) having a pH range from weakly basic to neutral to weaklyacidic; for example having a pH ranging from 10-3, or more preferably9-4, or more preferably 8-5, or even more preferably 7-6, depending onthe analyte of interest and the sorbent chemistry.

Any sample volume may be obtained as long as it is of sufficient volumeto be useful in the methods disclosed herein. In aspects of thisembodiment, a sample volume may be e.g., about 10 μL, about 25 μL, about50 μL, about 75 μL, about 100 μL, about 125 μL, about 150 μL, about 175μL, about 200 μL, about 225 μL, about 250 μL, about 275 μL, about 300μL, about 325 μL, about 350 μL, about 375 μL, about 400 μL, about 425μL, about 450 μL, about 475 μL, or about 500 μL. In other aspects ofthis embodiment, a sample volume may be e.g., at least 10 μL, at least25 μL, at least 50 μL, at least 75 μL, at least 100 μL, at least 125 μL,at least 150 μL, at least 175 μL, at least 200 μL, at least 225 μL, atleast 250 μL, at least 275 μL, at least 300 μL, at least 325 μL, atleast 350 μL, at least 375 μL, at least 400 μL, at least 425 μL, atleast 450 μL, at least 475 μL, or at least 500 μL. In yet other aspectsof this embodiment, a sample volume may be e.g., at most 10 μL, at most25 μL, at most 50 μL, at most 75 μL, at most 100 μL, at most 125 μL, atmost 150 μL, at most 175 μL, at most 200 μL, at most 225 μL, at most 250μL, at most 275 μL, at most 300 μL, at most 325 μL, at most 350 μL, atmost 375 μL, at most 400 μL, at most 425 μL, at most 450 μL, at most 475μL, or at most 500 μL. In still other aspects of this embodiment, asample volume may be between e.g., about 10 μL and about at most 100 μL,about 10 μL and about at most 200 μL, about 10 μL and about at most 300μL, about 10 μL and about at most 400 μL, about 10 μL and about at most500 μL, about 10 μL and about at most 600 μL, about 10 μL and about atmost 700 μL, about 10 μL and about at most 800 μL, about 10 μL and aboutat most 900 μL, about 10 μL and about at most 1000 μL, about 50 μL andabout at most 100 μL, about 50 μL and about at most 200 μL, about 50 μLand about at most 300 μL, about 50 μL and about at most 400 μL, about 50μL and about at most 500 μL, about 50 μL and about at most 600 μL, about50 μL and about at most 700 μL, about 50 μL and about at most 800 μL,about 50 μL and about at most 900 μL, about 50 μL and about at most 1000μL, about 100 μL and about at most 200 μL, about 100 μL and about atmost 300 μL, about 100 μL and about at most 400 μL, about 100 μL andabout at most 500 μL, about 100 μL and about at most 600 μL, about 100μL and about at most 700 μL, about 100 μL and about at most 800 μL,about 100 μL and about at most 900 μL, or about 100 μL and about at most1000 μL.

Purification

A test sample disclosed herein may be purified. As used herein, theterms “purified”, “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedparent or daughter ions of the selected analyte by mass spectrometry.Relative reduction as this term is used does not require that anysubstance, present with the analyte of interest in the material to bepurified, is entirely removed by purification. When detecting someanalytes (particularly drugs) in a urine sample, hydrolysis may benecessary to remove the glucuronide bonding which prevents thesolubility and extraction of the analyte. This purification technique isusually performed by enzyme or acid hydrolysis of the urine.Alternatively, removing particulate matter (e.g., by centrifugation orfiltration), protein precipitation (optionally by a “protein crash”method) with or without phospholipid removal, may be useful purificationtechniques.

Purification may also be performed to create or make available reactiveamino or phenolic groups, suitable for the derivatization reaction.These methods include hydrolysis of esters or amines, or acid hydrolysisof sugars.

Such purification by pre-processing is not limited, but serves toprepare the sample for extraction with one or more of solid phaseextraction, supported liquid extraction (SLP), and liquid liquidextraction (LLP).

Solid Phase Extraction

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis.

SPE using an ion exchange extraction procedure is applied to extract theanalytes of interest from the sample. Such analyte can be ionized undercertain ranges of pH of a buffer. SPE may be performed with a range ofcharacteristics suitable depending on the analyte. Analytes such asmonoamine neurotransmitters, or catecholamines, or metanephrines, oramino acids, or thyroid hormones, or carboxylic acids, maybe extracted,or retained, or purified, via ion exchange extraction based SPE. Morespecifically strong to weak cation exchange may be used. SPE using acation exchange is one example applied in the present methods ofextracting analyte of interest from a blood plasma sample. Weak cationexchange cartridges with a divinylbenzene- (DVB-) based polymer sorbentare particularly exemplified for the SPE of catecholamines andmetanephrines. SPE using a strong cation exchange extraction based SPEmay also be used to purify analytes from the blood plasma sample with anSPE cartridge filled with a DVB-based polymer sorbent via strongerelution solvent. Moreover, silica based carboxylic acid sorbents mayalso be useful to extract catecholamine and metanephrines from theplasma samples.

A strong cation exchange (sulfonic acid chemistry) sorbent either basedon silica or one or more polymers may also be useful to extract analytesin the SPE process. SPE with a strong cation exchange sorbent areparticularly exemplified for the SPE of thyroid hormones (T3/rT3/T4). Anamino acid analyte may be similarly extracted with a strong cationexchange sorbent, either polymer based, or silica based.

An anion exchange polymer may also be used to extract a carboxylic acidanalyte, or an amino acid analyte, or a sulfonic acid analyte, or aphosphonic acid analyte.

Particular columns of interest for use in the present methods to extractsteroids include the CEREX® PWCX, 1 mL Columns, 10 mg, 96/Pk (catalognumber 6750-0101R. For thyroid hormones the CEREX® PSCX, 1 mL Columns,10 mg, 96/Pk (catalog number 687-0101R) are suitable.

In yet another embodiment, a reverse phase SPE column or cartridge maybe used to extract, or purify, or retain the analyte of choice when theanalyte does not have an amino group, such as vitamin d its derivativesand metabolites, estrogen hormones, or cannabinoids, or flavonoids. Thesorbent of choice during this SPE/purification may include alkyl boundedsilica (C4, C8, C12, and C18), cyano bounded silica, phenyl boundedsilica, or biphenyl bounded silica. Particular columns of interestinclude Trace-N® 1 cc columns (a hydrophobic polymer/weak anion exchangecolumn, 10 um particle size, 300 A, from SPE Ware).

Sizes of the columns may range, but in particular, a column volume maypreferably range from 50 uL to 3000 uL, with a sorbent loading between100 ug to 50 mg. In another embodiment, the more preferred column sizeranges from 100 uL to 2000 uL with a sorbent loading between 1 mg to 20mg. In another embodiment, the more preferred column size ranges from200 uL to 1000 uL with a sorbent loading between 2 mg to 10 mg. Shapeand size of the SPE columns (cartridges) may be varied to fit a specificplatform.

The particle size of the sorbent may further assist in the separation ofthe analyte of interest. In aspects of this embodiment, a particle sizeof a sorbent may have a mean diameter of, e.g., about 0.5 μm, about 1μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm,about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about55 μm, about 60 μm, about 65 μm, about 70 μm, or about 75 μm. In otheraspects of this embodiment, a particle size of a sorbent may have a meandiameter of, e.g., at least 0.5 μm, at least 1 μm, at least 5 μm, atleast 10 μm, at least 15 μm, at least 20 μm, at least 25 μm, at least 30μm, at least 35 μm, at least 40 μm, at least 45 μm, at least 50 μm, atleast 55 μm, at least 60 μm, at least 65 μm, at least 70 μm, or at least75 μm. In yet other aspects of this embodiment, a particle size of asorbent may have a mean diameter of, e.g., at most 0.5 μm, at most 1 μm,at most 5 μm, at most 10 μm, at most 15 μm, at most 20 μm, at most 25μm, at most 30 μm, at most 35 μm, at most 40 μm, at most 45 μm, at most50 μm, at most 55 μm, at most 60 μm, at most 65 μm, at most 70 μm, or atmost 75 μm. In still other aspects of this embodiment, a particle sizeof a sorbent may have a mean diameter in the range of, e.g., about 0.5μm to about 10 μm, about 0.5 μm to about 20 μm, about 0.5 μm to about 30μm, about 0.5 μm to about 40 μm, about 0.5 μm to about 50 μm, about 0.5μm to about 60 μm, about 0.5 μm to about 70 μm, about 0.5 μm to about 80μm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about 1 μm toabout 30 μm, about 1 μm to about 40 μm, about 1 μm to about 50 μm, about1 μm to about 60 μm, about 1 μm to about 70 μm, about 1 μm to about 80μm, about 5 μm to about 10 μm, about 5 μm to about 20 μm, about 5 μm toabout 30 μm, about 5 μm to about 40 μm, about 5 μm to about 50 μm, about5 μm to about 60 μm, about 5 μm to about 70 μm, about 5 μm to about 80μm, about 10 μm to about 20 μm, about 10 μm to about 30 μm, about 10 μmto about 40 μm, about 10 μm to about 50 μm, about 10 μm to about 60 μm,about 10 μm to about 70 μm, or about 10 μm to about 80 μm.

A sample may be loaded on the SPE column with a loading solvent, or aloading buffer. The loading solvent may be deionized water, or a pHbuffered aqueous solution, or an organic solvent, or a mixture oforganic solvents, or a mixture of an organic solvent and deionizedwater, or a mixture of organic solvents with deionized water, or a pHbuffered aqueous solution mixed with an organic solvent or a mixture oforganic solvents. The pH buffered aqueous solution may be a phosphatebuffered saline (PBS), or a phosphate buffer, or a carbonate buffer, ora succinate buffer, or a tartrate buffer, or a citric buffer, or aformic buffer, or an acetic buffer, or another commonly used buffersolution in a typical biochemical lab, or a mixture of any of the two,or more of the following, a phosphate buffer, or a carbonate buffer, oran acetic buffer, or a formic buffer, or a citric buffer, or a succinatebuffer, or a tartrate buffer, or, with a pH range from weakly basic toneutral to weakly acidic; for example having a pH ranging from 10-3, ormore preferably 9-4, or more preferably 8-5, or even more preferably7-6, at a concentration range from 0.1 mM to 100 mM, or more preferably0.5 mM to 50 mM, or more preferably 1 mM to 25 mM, or more preferably 5mM to 10 mM. An organic solvent may be selected from acetonitrile, oracetone, or 1,4-dioxane, or DMF, or tetrahydrofuran (THF), ordiethylether, or ethyl acetate, or methyl acetate, or ethyl formate,methyl formate, or a mixture of thereof. In one embodiment, the sampleis loaded onto the column in a QUANTISIL® extraction and/or transferbuffer (Immunalysis Corporation, Pomona, Calif.).

Upon loading the sample to the SPE column, the fluid is allowed to passthrough the sorbent, via gravity, or a vacuum pulling through a vacuummanifold, or a nitrogen or inert gas pressure push through a positivepressure manifold, with or without an air drying process. The sampleloaded cartridge may be further washed with a solvent. The selection ofthe washing solvent may be deionized water, or a pH buffered aqueoussolution, or organic solvent, or a mixture of organic solvents, or amixture of organic solvents with an aqueous buffer. An organic solventmay be acetonitrile, or methanol, or ethanol, or isopropanol, orbutanol, or diethyl ether, or acetone, or 1,4-dioxane, or THF, or DMF,ethyl acetate, or methyl acetate, or ethyl formate, methyl formate, or amixture of any of the above solvents.

Upon loading and washing, the loaded cartridge may be treated with anelution fluid directly, or dried first via a stream of, dry nitrogen, oranother dry inner gas, passing through the cartridge.

Derivatization

Aspects of the present specification disclose, in part, a method ofderivatization of the analyte of interest using a derivatizing agent.The analyte of interest reacts with a derivatizing agent to provide aderivative of the analyte. Derivatization may be in situ. Thisderivative displays an improved HPLC behavior, significantly improvedtandem MS/MS sensitivity, and makes lipophilic analytes morehydrophilic. For instance the present method unexpectedly providesnearly, or over 1000-fold improvement in detection limitation and/orquantification sensitivity when quantifying vitamin D, cannabinoids,estrogen and other analytes from blood serum samples, by using LC-tandemMS/MS technologies.

A derivatizing reagent or derivatization reagent disclosed hereinincludes a derivatizing agent and a suitable solvent. The derivatizingagent is a compound that can react with a primary amino group, and/or aphenolic hydroxy group, and/or a primary alcohol (hydroxyl) group,and/or a aryl or alkyl thiol group, a cis-diene group, a carboxylic acidgroup, a ketone group, or an aldehyde group, present in an analytedisclosed herein. A derivatizing agent includes a crown-ether, aconnector, and a derivatizing functional group.

In one embodiment, the derivatized structure has a crown-ether component(CE), a connector or linker component (L), a derivatization (or analytebinding) functional group (FG), and an analyte. Formula 1 provides thegeneral structure of the claimed derivatizing agent:

CE-L-FG  Formula 1

The crown ether CE, may attach to or be fused to the linker L, which isconnected to the derivatizing functional group FG, which may covalentlyattach to certain selected analytes. One particular example of thederivatizing agent is shown in FIG. 1, demonstrating a specificembodiment of a derivatizing agent (100), having a crown-ether component(i.e., a sensitivity booster) (101), bound to a linker (also known asthe connector) (102), link to a functional group, which will in turncovalently link to the analyte (the derivatization functionalgroup)(103).

Crown ethers of interest include any crown ether and variants thereof.In one aspect the crown ether is optionally fused to one or morecyclohexyl or aryl groups, optionally where one or more oxygens in thecrown is substituted with a heteroatom, such as nitrogen (i.e.,aza-crown ethers) or sulfur (thia-crown ethers). In one embodiment, thecrown ether includes a 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 membered ring. In one embodiment, thecrown ether ranges from 12 crown 4 to 24 crown 8. In one aspect, the“crown” of the crown ether is 12 Crown 4, 15 Crown 5, 16 crown 4, 18Crown 6, 21 Crown 7, or 24 Crown 8.

The crown ether may have a lariat structure as well. The lariatstructure may include a C1-C12 alkyl group (linear, cyclic, and/orbranched), optionally substituted or interrupted. For instance, thelariat structure may serve as the linker or connector or linker betweenthe crown ether and the bound analyte.

The linker or connector is bound to the crown ether by a simple covalentbond. In one embodiment, the linker or connector may be a C₁-C₁₂ linear,branched or cyclic alkyl group. In one aspect, the linker or connectormay be a C₁-C₆ linear, branched or cyclic alkyl. In another aspect, thelinker or connector is fused to the crown ether forming a six-memberedphenolic ring.

The derivatization functional group that binds the analyte may include,without limitation, an acylating group,4-Phenyl-1,2,4-triazolin-3,5-dione (PTAD), 1,2,4-traizoline-3,5-dione(TAD), Alkoxylamines, hydrazides, alcohols, or amines. In oneembodiment, the derivatization functional group may be any acylatinggroup as in Formula 2. Acylating reagents may include acyl chlorides orother acyl halides. Derivatizing agents may also fluoresce orparticipate in a colorimetric reaction to assist with the detection ofthe bound analyte.

The acylation group in the derivatizing agent is useful for analyteswith a primary and secondary amine, aliphatic hydroxyl, or phenolichydroxyl group. Such as monoamines, amino acids, estrogen hormones, THC,its metabolites and analogs, such as HU210, and etc. In one embodiment,the crown ether derivatizing agent with an acylating functional groupincludes, without limitation, any of the structures of Group I:

In one aspect, the crown ether derivatizing agent is MB338.

Crown ether containing derivatizing agents for derivatizing a cis-dienecontaining analyte include, without limitation, crown ethers bound to4-Phenyl-1,2,4-triazolin-3,5-dione (PTAD) or 1,2,4-traizoline-3,5-dione(TAD) either directly, or by a linking/connecting group or lariat. Suchderivatizing agents include, without limitation, any of the structuresof Group II:

In one aspect, the derivatizing agent is MB409. In another embodiment,either the crown ether or the analyte may be further bound or connectedto a 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) moiety, or a1,2,4-triazoline-3,5-dione (TAD) moiety. For instance, when bound tovitamin D, PTAD creates two epimers by reacting with the s-cis-dienemoiety of vitamin D.

Crown ether derivatising agents suitable for derivatizing aldehydes andketones (such as testosterone and many of the ketone and aldehydecontaining steroids) include without limitation, crown ethers bound toalkoxylamines and hydrazides. In one embodiment, the derivatizing agenthaving a crown ether bound to an alkoxylamine or hydrazide, includes,without limitation any of the structures of Group III:

Crown ether derivatizing agents suitable for derivatizing carboxylicacids (such as aliphatic acids, biotin, monomethyl malonic acid (MMA),etc.) include without limitation, crown ethers bound to alcohols oramines. In one embodiment, the derivatising agent is a crown ether boundto an alcohol or amine, which includes, without limitation any of thestructures of Group IV:

The crown ethers serve as a mass spectrometry sensitivity enhancer bytrapping a cation. Cations of interest include, without limitation,NH4+, H3O+, MeNH3+, Na+, K+, or Ca2+. Different crown ethers havedifferent affinities to different cations based on the size and shape ofthe crown ether.

In situ derivatization is performed either just before elution, duringelution, or right after elution, or from just before the elutioncontinued until after the elution, from the solid phase extractioncolumn.

The elution may be performed with a high pH elution solution. As usedherein, “high pH” includes elution solutions having a basic pH. In anaspect of this embodiment, an elution solution may have a pH of, e.g.,about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11,about 11.5, about 12, about 12.5, or about 13. In other aspect of thisembodiment, an elution solution may have a pH of, e.g., at least 8, atleast 8.5, at least 9, at least 9.5, at least 10, at least 10.5, atleast 11, at least 11.5, at least 12, at least 12.5, or at least 13. Inyet other aspect of this embodiment, an elution solution may have a pHof, e.g., at most 8, at most 8.5, at most 9, at most 9.5, at most 10, atmost 10.5, at most 11, at most 11.5, at most 12, at most 12.5, or atmost 13. In yet other aspect of this embodiment, an elution solution mayhave a pH in the range of, e.g., about 8 to about 9, about 8 to about9.5, about 8 to about 10, about 8 to about 10.5, about 8 to about 11,about 8 to about 11.5, about 8 to about 12, about 8 to about 12.5, about8 to about 13, about 8.5 to about 9, about 8.5 to about 9.5, about 8.5to about 10, about 8.5 to about 10.5, about 8.5 to about 11, about 8.5to about 11.5, about 8.5 to about 12, about 8.5 to about 12.5, about 8.5to about 13, about 9 to about 9.5, about 9 to about 10, about 9 to about10.5, about 9 to about 11, about 9 to about 11.5, about 9 to about 12,about 9 to about 12.5, about 9 to about 13, about 9.5 to about 10, about9.5 to about 10.5, about 9.5 to about 11, about 9.5 to about 11.5, about9.5 to about 12, about 9.5 to about 12.5, about 9.5 to about 13, about10 to about 10.5, about 10 to about 11, about 10 to about 11.5, about 10to about 12, about 10 to about 12.5, about 10 to about 13, about 10.5 toabout 11, about 10.5 to about 11.5, about 10.5 to about 12, about 10.5to about 12.5, about 10.5 to about 13, about 11 to about 11.5, about 11to about 12, about 11 to about 12.5, or about 11 to about 13.

An elution solution disclosed herein may be buffered using any bufferhaving an alkaline buffering capacity. In aspects of this embodiment, anelution solution disclosed herein may be buffered using one or a mixtureof the organic or inorganic buffering agents, e.g., POPSO, TEA,phosphate. In other aspects of this embodiment, an elution solutiondisclosed herein may be buffered using, e.g., a trialkyl-ammonium buffercomprising, e.g., trialkylammonium bicarbonate, trialkylammonium borate,trialkylammonium carbonate, or a trialkylammonium phosphate; a cesiumbuffer comprising, e.g., cesium bicarbonate, cesium borate, cesiumcarbonate, cesium hydroxide, or dibasic cesium phosphate, or tribasiccesium phosphate; a potassium buffer comprising, e.g., potassiumbicarbonate, potassium borate, potassium carbonate, potassium hydroxide,or dibasic potassium phosphate, or tripotassium phosphate; a sodiumbuffer comprising, e.g., sodium bicarbonate, sodium borate, sodiumcarbonate, dibasic sodium phosphate, tribasic sodium phosphate, sodiumhydroxide, or sodium tetraborate; a tetraalkylammonium buffer,comprising, e.g., tetraalkylammonium bicarbonate, tetraalkylammoniumborate, tetraalkylammonium carbonate, or a tetraalkylammonium phosphate,in water, with or without the use of one of more of the followingorganic co-solvents such as acetonitrile, or acetone, or tetrohydrofuran(THF), or 1,4-dioxane, or dimethylformamide (DMF), or N-methylpyrrolidone (NMP), or dimethyl sulfoxide (DMSO), orhexamethylphosphoramide (HMPA), or diethyl ether, or isopropyl alcohol(IPA), or t-butanol, or 2-butanol, etc, in a desired ratio, such asat/below/or above, 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%,or 40%, or 45%, or 50%, or 55%, or 60%, or 70%, or 75%, or 80%, or 85%,or 90%, or 95%, of organic in water.

The amount of buffer used in an elution solution may be anyconcentration that can effectively maintain the alkaline bufferingcapacity of the buffer. In aspects of this embodiment, an effectiveconcentration of buffer may be, e.g., about 1.0 mM, about 5.0 mM, about10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM,about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about800 mM, or about 900 mM, or about 1 M. In other aspects of thisembodiment, an effective concentration of buffer may be, e.g., at least1.0 mM, at least 5.0 mM, at least 10 mM, at least 20 mM, at least 30 mM,at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least80 mM, at least 90 mM, at least 100 mM, at least 200 mM, at least 300mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM,at least 800 mM, or at least 900 mM, or at least 1 M. In yet otheraspects of this embodiment, an effective concentration of buffer may be,e.g., at most 1.0 mM, at most 5.0 mM, at most 10 mM, at most 20 mM, atmost 30 mM, at most 40 mM, at most 50 mM, at most 60 mM, at most 70 mM,at most 80 mM, at most 90 mM, at most 100 mM, at most 200 mM, at most300 mM, at most 400 mM, at most 500 mM, at most 600 mM, at most 700 mM,at most 800 mM, or at most 900 mM, or at most 1 M.

In still other aspects of this embodiment, an effective concentration ofelution buffer may be in the range of, e.g., about 0.1 mM to about 10mM, about 0.1 mM to about 25 mM, about 0.1 mM to about 50 mM, about 0.1mM to about 75 mM, about 0.1 mM to about 100 mM, about 0.1 mM to about200 mM, about 0.1 mM to about 300 mM, about 0.1 mM to about 400 mM,about 0.1 mM to about 500 mM, about 0.1 mM to about 1000 mM, about 1 mMto about 10 mM, about 1 mM to about 25 mM, about 1 mM to about 50 mM,about 1 mM to about 75 mM, about 1 mM to about 100 mM, about 1 mM toabout 200 mM, about 1 mM to about 300 mM, about 1 mM to about 400 mM,about 1 mM to about 500 mM, about 1 mM to about 1000 mM, about 5 mM toabout 25 mM, about 5 mM to about 50 mM, about 5 mM to about 75 mM, about5 mM to about 100 mM, about 5 mM to about 200 mM, about 5 mM to about300 mM, about 5 mM to about 400 mM, about 5 mM to about 500 mM, about 5mM to about 1000 mM, about 10 mM to about 25 mM, about 10 mM to about 50mM, about 10 mM to about 75 mM, about 10 mM to about 100 mM, about 10 mMto about 200 mM, about 10 mM to about 300 mM, about 10 mM to about 400mM, about 10 mM to about 500 mM, about 10 mM to about 1000 mM, about 25mM to about 50 mM, about 25 mM to about 75 mM, about 25 mM to about 100mM, about 25 mM to about 200 mM, about 25 mM to about 300 mM, about 25mM to about 400 mM, about 25 mM to about 500 mM, about 25 mM to about1000 mM, about 50 mM to about 75 mM, about 50 mM to about 100 mM, about50 mM to about 200 mM, about 50 mM to about 300 mM, about 50 mM to about400 mM, about 50 mM to about 500 mM, about 50 mM to about 1000 mM, about75 mM to about 100 mM, about 75 mM to about 200 mM, about 75 mM to about300 mM, about 75 mM to about 400 mM, about 75 mM to about 500 mM, about75 mM to about 1000 mM, about 100 mM to about 150 mM, about 100 mM toabout 200 mM, about 100 mM to about 300 mM, about 100 mM to about 400mM, about 100 mM to about 500 mM, about 100 mM to about 1000 mM, about200 mM to about 300 mM, about 200 mM to about 400 mM, about 200 mM toabout 500 mM, about 200 mM to about 1000 mM, about 250 mM to about 300mM, about 250 mM to about 400 mM, about 250 mM to about 500 mM, or about250 mM to about 1000 mM. In another embodiment, an effectiveconcentration of the elution buffer may be in the range of about 5 mM toabout 250 mM.

An elution solution disclosed herein may comprise a derivatizing agentdisclosed herein. The amount of derivatizing agent added to an elutionsolution disclosed herein is an amount in sufficient access to enable acomplete derivatization of the analyte of interest for subsequentdetection. The derivatizing agent may be mixed with the elution bufferprior to the elution as in an in situ derivatization process, it mayalso be added to the eluent right after the elution to fashion a postelution derivatization process.

In one embodiment, the simultaneous elution and derivatization of theanalyte may be accomplished by applying to the analyte bound to a solidsorbent support matrix an elution buffer which includes a derivatizingagent. Upon application of the elution solution, a derivatizationreaction occurs that converts the analyte to its derivative and then thederivative is eluted off the sorbent matrix at the same time as theelution of the analyte from the solid support matrix.

The derivatization reaction may be conducted under any conditionsuitable for the binding of the analyte. In one embodiment, aderivatization reaction is performed under temperature conditionssuitable for the attachment of the derivatizing agent to the analyte. Inaspects of this embodiment, a derivatization reaction may be performedat a temperature range from about or above 0° C. to about or below 100°C., more preferably from about 5° C. to about 90° C., more preferablyfrom 10° C. to 80° C., more preferably from 15° C. to 70° C., morepreferably from 18° C. to 60° C., more preferably from 18° C. to 40° C.

A derivatization reaction is performed under time conditions suitablefor the attachment of the derivatizing agent to the analyte. In aspectsof this embodiment, the derivatization reaction is performed at durationrange from about 1 minute to about 24 hours, more preferably from about3 minutes to about 5 hours, more preferably from about 5 minutes toabout 60 minutes, more preferably from about 10 minutes to about 45minutes. In one aspect the reaction ranges from about 15 minutes toabout 30 minutes.

Of course, changing the temperature may change the reaction time. Forinstance, if the reaction is heated, for example, to 40° C., reactiontime may be shortened.

The derivatization reaction may be quenched by addition of a bufferedsolution, which contains one or more of buffer agents. In oneembodiment, the buffer is an ammonium buffer, such as ammonium formate,or ammonium acetate, or ammonium carbonate, or triammonium phosphate, orammonium sulfate, or ammonium borate, ammonium hydroxide, ammoniumchloride, ammonium sulfate, ammonium bicarbonate, ammonium bisulfate,bisammonium phosphate, etc. In another embodiment, the quenching reagentmay also be a buffered □-amino acid solution, such as glycine, alanine,or a buffered □-alanine, or a buffered primary amine solution such as ina concentration range from 1 mM to 500 mM, in water, or a mixed solventof water and an organic solvent, such as an alcohol, e.g., methanol, orethanol, or propanol, or butanol, or glycol, or glycerol, or etc., oracetonitrile, or acetone, or ether, or THF, or 1,4-dioxanes, or DMF, orNMP, or DMSO, or HMPA. The quenching process neutralizes most of anyexcess derivatizing reagent that may remain in the eluent solution andalso stabilizes the product by lowering the pH of the reaction mixture.In aspects of this embodiment, the pH of the eluent containing thederivatized analyte may be lowered to a range from about 4 to about 9.5.

Chromatography Analysis

After quenching, the reaction mixture containing derivatized analyte maythen be directly analyzed for the presence of the analyte of interest.Such analysis may be either qualitative or quantitative in nature. Inaspects of this embodiment, eluent containing derivatized analyte may bedirectly analyzed for the presence of the analyte of interest using,e.g., chromatography and/or mass spectroscopic detection.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), ultra-high pressure liquidchromatography (UHPLC), and turbulent flow liquid chromatography (TFLC)(sometimes known as high turbulence liquid chromatography) (HTLC) orhigh throughput liquid chromatography, or nano-flow liquidchromatography, or nano LC

As used herein, the term “high performance liquid chromatography” or“HPLC” refers to liquid chromatography in which the degree of separationis increased by forcing the mobile phase under pressure through astationary phase, typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography (HTLC)or high throughput liquid chromatography) refers to a form ofchromatography that utilizes turbulent flow of the material beingassayed through the column packing as the basis for performing theseparation. TFLC has been applied in the preparation of samplescontaining two unnamed drugs prior to analysis by mass spectrometry.See, e.g., Zimmer et al., J Chromatogr A 854: 23-35 (1999); see also,U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, whichfurther explain TFLC. Persons of ordinary skill in the art understand“turbulent flow”. When fluid flows slowly and smoothly, the flow iscalled “laminar flow”. For example, fluid moving through an HPLC columnat low flow rates is laminar. In laminar flow the motion of theparticles of fluid is orderly with particles moving generally instraight lines. At faster velocities, the inertia of the water overcomesfluid frictional forces and turbulent flow results. Fluid not in contactwith the irregular boundary “outruns” that which is slowed by frictionor deflected by an uneven surface. When a fluid is flowing turbulently,it flows in eddies and whirls (or vortices), with more “drag” than whenthe flow is laminar. Many references are available for assisting indetermining when fluid flow is laminar or turbulent (e.g., TurbulentFlow Analysis Measurement and Prediction, P. S. Bernard & J. M. Wallace,John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow, JeanMathieu & Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm. As used in this context, the term“about” means±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%.

Certain methods of liquid chromatography, including HPLC, rely onrelatively slow, laminar flow technology. Traditional HPLC analysisrelies on column packing in which laminar flow of the sample through thecolumn is the basis for separation of the analyte of interest from thesample. The skilled artisan will understand that separation in suchcolumns is a diffusional process and may select HPLC instruments andcolumns that are suitable for use with the analytes of interest. Thechromatographic column typically includes a medium (i.e., a packingmaterial) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlesinclude a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the chemical moieties. One suitablebonded surface is a hydrophobic bonded surface such as an alkyl bonded,a cyano bonded, or a pentafluorophenylpropyl (F5) surface, orphenyl/bonded, or biphenyl bonded surface. Alkyl bonded surfaces mayinclude C-4, C-8, C-12, or C-18 bonded alkyl groups. In preferredembodiments, the column is a C-18 column. The chromatographic columnincludes an inlet port for receiving a sample directly or indirectlyfrom a solid-phase extraction or HTLC column and an outlet port fordischarging an effluent that includes the fractionated sample.

In certain embodiments, an analyte may be enriched in a sample byapplying a sample to a column under conditions where the analyte ofinterest is reversibly retained by the column packing material, whileone or more other materials are not retained. In these embodiments, afirst mobile phase condition can be employed where the analyte ofinterest is retained by the column, and a second mobile phase conditioncan subsequently be employed to remove retained material from thecolumn, once the non-retained materials are washed through.Alternatively, an analyte may be enriched in a sample by applying asample to a column under mobile phase conditions where the analyte ofinterest elutes at a differential rate in comparison to one or moreother materials. Such procedures may enrich the amount of one or moreanalytes of interest relative to one or more other components of thesample. In another embodiment, the reaction mixture of the analyte maybe first loaded onto a guard column with a weak solvent to retain thedesired analyte product on the guard column, then an LC elution solventis to carry the substrate onto the analytical column for separation andanalysis.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytypic (i.e.mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In one preferred embodiment, HPLC is conducted with a hydrophobic columnchromatographic system. In certain preferred embodiments, a C18analytical column (e.g., a TARGA® C18, 3 μm 50×2.1, or equivalent) isused. In certain preferred embodiments, HPLC are performed using HPLCGrade 5.0 mM ammonium formate with 0.1% formic acid at a pH of 3.0 and0.1% formic acid in acetonitrile as the mobile phases.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, the solid phase extraction may be used in a highthroughput platform for enrichment of the derivatized analyte ofinterest prior to mass spectrometry. In such embodiments, samples may beextracted using a high throughput SPE cartridge, or a guard column whichcaptures the derivatized analyte, then eluted onto an analytical HPLCcolumn, such as a C-18 column, prior to mass spectrometry (MS) analysis.Because the steps involved in these chromatography procedures may belinked in an automated fashion, the requirement for operator involvementduring the purification of the analyte can be minimized. This featuremay result in savings of time and costs, and eliminate or reduce theopportunity for an operator error.

Direct Quantification

Direct quantification is “inline” or “on-line” use of the extracted andderivatized analyte for quantification. As used herein, the term“on-line” or “inline”, for example as used in “on-line automatedfashion” or “on-line extraction” refers to a procedure performed withoutthe need for operator intervention. In contrast, the term “off-line” asused herein refers to a procedure requiring manual intervention of anoperator. Thus, if samples are subjected to precipitation, and thesupernatants are then manually loaded into an autosampler, theprecipitation and loading steps are off-line from the subsequent steps.In various embodiments of the methods, one or more steps may beperformed in an on-line automated fashion.

As used herein, the term “sample injection” refers to introducing analiquot of a single sample into an analytical instrument, for example amass spectrometer. This introduction may occur directly or indirectly.An indirect sample injection may be accomplished, for example, byinjecting an aliquot of a sample into a HPLC column that is connected toa mass spectrometer in an on-line fashion.

As used herein, the term “same sample injection” with respect tomultiple analyte analysis by mass spectrometry means that the molecularions for two or more different analytes are determined essentiallysimultaneously by measuring molecular ions for the different analytesfrom the same (i.e. identical) sample injection.

Mass Spectrometry

In various embodiments, the analytes of interest present in a testsample may be ionized by any method known to the skilled artisan. Massspectrometry is performed using a mass spectrometer, which includes anion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. For example ionization of the sample maybe performed by electron ionization, chemical ionization, electrosprayionization (ESI), photon ionization, atmospheric pressure chemicalionization (APCI), photoionization, atmospheric pressure photoionization(APPI), fast atom bombardment (FAB), liquid secondary ionization (LSI),matrix assisted laser desorption ionization (MALDI), field ionization,field desorption, thermospray/plasmaspray ionization, surface enhancedlaser desorption ionization (SELDI), inductively coupled plasma (ICP)and particle beam ionization. The skilled artisan will understand thatthe choice of ionization method may be determined based on the analyteto be measured, type of sample, the type of detector, the choice ofpositive versus negative mode, etc.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; andMerchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which may be heated to prevent condensation and to facilitatesolvent evaporation. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased. In one embodiment, the detection is performed after ESI.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counter flow of dry andpreheated N2 gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “desorption” refers to translocation of ananalyte from a liquid surface and/or the entry of an analyte into agaseous phase. Laser desorption thermal desorption is a techniquewherein a sample containing the analyte is thermally desorbed into thegas phase by a laser pulse. The laser hits the back of a specially made96-well plate with a metal base. The laser pulse heats the base and theheat causes the sample to transfer into the gas phase. The gas phasesample is then drawn into the mass spectrometer.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

The ions may be detected using several detection modes. For example,selected ions may be detected, i.e. using a selective ion monitoringmode (SIM), or alternatively, ions may be detected using a scanningmode, e.g., multiple reaction monitoring (MRM) or selected reactionmonitoring (SRM). Preferably, the mass-to-charge ratio is determinedusing a quadrupole analyzer. For example, in a “quadrupole” or“quadrupole ion trap” instrument, ions in an oscillating radio frequencyfield experience a force proportional to the DC potential appliedbetween electrodes, the amplitude of the RF signal, and the mass/chargeratio. The voltage and amplitude may be selected so that only ionshaving a particular mass/charge ratio travel the length of thequadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS filter anddetector (quadrupole). By careful selection of precursor ions, only ionsproduced by certain analytes are passed to the fragmentation chamber,where collisions with atoms of an inert gas produce the fragment ions.Because both the precursor and fragment ions are produced in areproducible fashion under a given set of ionization/fragmentationconditions, the MS/MS technique may provide an extremely powerfulanalytical tool. For example, the combination offiltration/fragmentation may be used to eliminate interferingsubstances, and may be particularly useful in complex samples, such asbiological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g. m/z: 5-1250 for API 5000) The results of ananalyte assay, that is, a mass spectrum, may be related to the amount ofthe analyte in the original sample by numerous methods known in the art.For example, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, molecular standards may be run withthe samples, and a standard curve constructed based on ions generatedfrom those standards. Using such a standard curve, the relativeabundance of a given ion may be converted into an absolute amount of theoriginal molecule. In certain preferred embodiments, one or moreinternal standards may be used to generate standard curves forcalculating the quantity of the analytes of interest. Methods ofgenerating and using such standard curves are well known in the art andone of ordinary skill is capable of selecting appropriate internalstandards. For example, an isotopically labeled analyte may be used asan internal standard; in certain preferred embodiments, D6-25 OH vitaminD₃, D6-25-OH Vitamin D₂, D6-1,25(OH)₂ vitamin D₃, D6-1,25(OH)₂ vitaminD₂ etc., may be used as internal standards. Numerous other methods forrelating the amount of an ion to the amount of the original moleculewill be well known to those of ordinary skill in the art.

In particularly preferred embodiments, the analytes of interest arequantified in a sample using MS/MS as follows. One or more of theanalytes of interest in samples are first filtered through and elutedfrom a solid phase extraction column at a high pH in the presence ofFMOC-CI or a variant thereof. The resulting eluent is then subjected toliquid chromatography, preferably HPLC. The flow mobile phase from thechromatographic column enters the heated ESI probe of an MS/MS analyzerand the analytes ionized. The ions, e.g. precursor ions, pass throughthe orifice of the instrument and enter the first quadrupole.Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing selection ofions (i.e., selection of “precursor” and “fragment” ions in Q1 and Q3,respectively) based on their mass to charge ratio (m/z). Quadrupole 2(Q2) is the collision cell, where ions are fragmented. The firstquadrupole of the mass spectrometer (Q1) selects for molecules with themass to charge ratios the analytes of interest. Precursor ions with thecorrect mass/charge ratios are allowed to pass into the collisionchamber (Q2), while unwanted ions with any other mass/charge ratiocollide with the sides of the quadrupole and are eliminated. Precursorions entering Q2 collide with neutral argon gas molecules and fragment.This process is called collision activated dissociation (CAD), orcollision induced dissociation (CID). The fragment ions generated arepassed into quadrupole 3 (Q3), where the fragment ions are selectedwhile other ions are eliminated. During analysis of a single sample, Q1and/or Q3 may be adjusted such that mass/charge ratios of one or moreprecursor ion/fragment ion pairs specific to one specific analyte isfirst selected, followed at some later time by the selection ofmass/charge ratios of one or more precursor ion/fragment ion pairsspecific to a second specific analyte, optionally followed at some latertime by the selection of mass/charge ratios of one or more precursorion/fragment ion pairs specific to a third specific analyte and so on.In particularly preferred embodiments, mass to charge ratios ofprecursor/fragment ion pairs specific to epinephrine, mass to chargeratios of precursor/fragment ion pairs specific to norepinephrine, massto charge ratios of precursor/fragment ion pairs specific to the analyteare detected during analysis of a single sample, although the sequenceof detection may occur in any order.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of the analyte ofinterest that may be used for selection in quadrupole 3 (Q3).

In various embodiments, the analyte of interest is subjected to a massspectrometry for detection and quantification. A mass spectrometrytechnique may employ atmospheric pressure chemical ionization (APCI) orelectrospray ionization (ESI) to generate charged ions. The analyte ofinterest can present as a proton adduct or a protonated molecular ion,i.e. [M+H⁺] in the mobile phase. The analyte can also be shown theammonium adduct [M+NH₄ ⁺] as a molecular ion when abundant ammonium ionis present in the mobile phase, or other cation adduct whencorresponding cations are present in the mobile phase. Different adductsare also possible and can be recognized by the skilled artisan, and aregenerally shown by [M+A+H]⁺, where A is the adduct. The adducts may ormay not be solvated. During the ionization process, the molecular ionsare desorbed into the gaseous phase, and then focused into the massspectrometer for analysis and detection. See U.S. Pat. No. 6,692,971 formore information on APCI, as it is known to those of skill of the art.

MS analysis can be done with a single mass analyzer such as a singlequardrupole mass spectrometer (MS), or a tandem mass analyzer such as atriple quardrupole tandem mass spectrometer (MS/MS). In a tandem massspectrometry mode, the first mass filter or quardrupole (Q1) can betuned to select independently, one or more of the molecular ions of theanalyte of interest and internal standards of choice. The molecular ions(precursor ions) can undergo collision-induced dissociation (CID) atsecond quardrupole (Q2) to produce fragment or product ions. Thefragment ions can be detected and analyzed at the second mass filter atQ3. This process can be referred to as product optimization. The secondmass filter is then tuned to selectively monitor one or more of the mostabundant product ions produced from a particular molecular ion. Thistechnique is called multiple reaction monitoring (MRM).

MRM transitions of precursor-product ion pairs can be monitored for thehormones estrone, estradiol and 17β-estradiol-2,3,4-¹³C₃ with MB338 asfollows:

TABLE 1 MRM Transitions for Hormones Compound Polarity Precursor m/zProduct m/z Estrone MB338 Positive 626.46 338.8 Estrone MB338 Positive626.46 163 Estradiol MB338 Positive 628.56 339.1 Estradiol MB338Positive 628.56 162.9 Estriol MB338 Positive 644.28 339 Estriol MB338Positive 644.28 162.8 17β-estradiol-2,3,4-¹³C₃ Positive 631.399 339MB338 17β-estradiol-2,3,4-¹³C₃ Positive 631.399 163 MB33817β-estradiol-2,3,4-¹³C₃ Positive 631.399 107 MB338

Molecular ions [M+NH₄ ⁺] of crown ether derivatives of1,25-dihydroxy-vitamin D and its derivatives may be identified as MRMtransitions of precursor-product ion pair. Internal standards, such asdeuterated analytes, can be applied in the methods described herein. Inone embodiment, D6-25 OH vitamin D₃, D6-25-OH Vitamin D₂, D6-1,25(OH)₂vitamin D₃, D6-1,25(OH)₂ vitamin D₂ may be used.

Molecular ions [M+NH₄ ⁺] of crown-ether derivatives of THC or HU210 withMB338 are shown at below as MRM transitions of precursor-product ionpair:

TABLE 2 MRM Transitions for THC and HU210 Compound Polarity Precursorm/z Product m/z THC MB338 Positive 670 339 THC MB338 Positive 670 163THC MB338 Positive 670 107 THC d3 MB338 Positive 673 339 THC d3 MB338Positive 673 163 THC d3 MB338 Positive 673 107 HU210 MB338 Positive 742337 HU210 MB338 Positive 742 339 HU210 MB338 Positive 742 163

The methods disclosed herein can be evaluated by several parametersincluding, e.g., accuracy, precision, limit of detection (LOD), limitsof quantitation (LOQ), linear range, specificity, selectivity,linearity, ruggedness, and system suitability. The accuracy of a methodis the measure of exactness of an analytical method, or the closeness ofagreement between the measured value and the value that is accepted as aconventional true value or an accepted reference value. The precision ofa method is the degree of agreement among individual test results, whenthe procedure is applied repeatedly to multiple samplings of ahomogeneous sample. As such, precision evaluates 1) within assayvariability; 2) within-day variability (repeatability); and 3)between-day variability (intermediate precision); and 4) between-labvariability (reproducibility). Coefficient of variation (CV %) is aquantitative measure of precision expressed relative to the observed ortheoretical mean value. The limit of detection (LOD) of a method refersto the concentration of analyte which gives rise to a signal that issignificantly different from the negative control or blank andrepresents the lowest concentration of analyte that can be distinguishedfrom background.

The limits of quantitation (LOQ) are the lowest and the highestconcentrations of analyte in a sample that can be measured with anacceptable level of accuracy and precision. The lower limit ofquantitation refers to the lowest dose that a detection method canmeasure consistently from the background. The upper limit ofquantitation is the highest dose that a detection method can measureconsistently before saturation of the signal occurs. The linear range ofthe method is the area between the lower and the upper limits ofquantitation. The linear range is calculated by subtracting lower limitof quantitation from the upper limit of quantitation. As used herein,the term “signal to noise ratio for the lower asymptote” refers to thesignal detected in the method at the lower limit of detection divided bythe background signal. As used herein, the term “signal to noise ratiofor the upper asymptote” refers to the signal detected in the method atthe upper limit of detection divided by the background signal.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of body fluid. However, an amount also contemplatesa relative amount in comparison to another analyte amount. For example,an amount of analyte in a body fluid can be an amount which is greaterthan a control or normal level of analyte normally present.

The present methods, reagents, and kits may be used for thequantification or the detection of an analyte of interest.

Embodiments Section

Embodiment 1 is a method for determining the presence of one or moreanalytes in a test sample, the method comprising:

-   -   a) extraction and purification of the analytes from the test        sample with one or more of solid phase extraction, supported        liquid extraction (SLP), and liquid liquid extraction (LLP);    -   b) derivatization of the analytes with a crown-ether        derivatizing agent;    -   c) detection of the derivatized analytes using liquid        chromatography and/or mass spectrometry.

In an aspect of Embodiment 1, the analyte is a compound having a primaryamine or a phenolic hydroxyl group. In a further aspect, the analyte isa drug, a hormone, a signaling agent, an amino acid, or a pesticide. Ina particular aspect, the analyte is a monoamine neurotransmitterincluding vitamin D or one of its derivatives or metabolites, a sexhormone or one of its derivatives or metabolites, a cannabinoid or oneof its derivatives or metabolites, an opiate, opioid or one of itsderivatives or metabolites or an arylcyclohexylamine or one of itsderivatives or metabolites, an Amphetamine or one of its derivatives ormetabolites. For instance, the monoamine neurotransmitter is Histamine,Tryptamine, Serotonin, or Agmatine. Similarly the sex hormone or one ofits derivatives or metabolites is an estrogen. Further, the derivativeof vitamin D is 25-OH D₃, 25-OH D₂, 24,25-(OH)₂ D₃, 1,25-(OH)₂ D₃, and1,25-(OH)₂ D₂, Cholecalciferol, 25-Hydroxycholecalciferol,1α,25-Dihydroxycholecalciferol, Ergocalciferol,1α,25-Dihydroxyergocalciferol, 22,23-Dihydroergocalciferol,1α,24R,25-Trihydroxycholecalciferol, (6Z)-tacalciol, Tachysterol₃,Isovitamin D₃, Dihydrotachysterol₃.

In yet another aspect of Embodiment 1, the cannabinoid or one of itsderivatives or metabolites is a Cannabigerol-type (CBG) cannabinoid, aCannabichromene-type (CBC) cannabinoid, a Cannabidiol-type (CBD)cannabinoid, a Cannabinodiol-type (CBND) cannabinoid, aTetrahydrocannabinol-type (THC) cannabinoid, a Cannabinol-type (CBN)cannabinoid, a Cannabitriol-type (CBT) cannabinoid, a Cannabielsoin-type(CBE) cannabinoid, an Isocannabinoid, a Cannabicyclol-type (CBL)cannabinoid, a Cannabicitran-type (CBT) cannabinoid, or aCannabichromanone-type (CBCN) cannabinoid. Similarly, the opiate, theopioid or the derivative or metabolite of the opiate or opioid, ismorphine, oripavine, morphinone, hydromorphone, or oxymorphone. In aparticular aspect, the opiate, the opioid, or the derivative ormetabolite of the opiate or opioid is a benzylisoquinoline alkaloid, asemi-synthetic benzylisoquinoline alkaloid derivative, or an opioid.

In yet another aspect, the arylcyclohexylamine or one of its derivativesor metabolites is Tiletamine, 3-Methoxetamine (MXE), Methoxyketamine,N-Ethylnorletamine (Ethketamine), Amphetamine, Ephedrine, orMethamphetamine.

In an even further aspect, the Amphetamine or one of its derivatives ormetabolites is Amphetamine (itself), methamphetamine, ephedrine,cathinone, 3,4-methylenedioxy-N-methylamphetamine (MDMA, “Ecstasy”), and2,5-Dimethoxy-4-methylamphetamine (DOM, or “STP”).

In a particular aspect of Embodiment 1, the solid phase extraction isperformed with an ion exchange column or cartridge. For instance in oneparticular aspect, the ion exchange column is a cation exchange column.For instance, the cation exchange column may be a weak cation exchangecolumn. In another particular aspect, the ion exchange column is ananion exchange column. In yet another aspect, the solid phase extractionis performed with a reverse phase silica column or cartridge. Forinstance, the reverse phase silica may be an alkyl bounded (C4, C8, C12,or C18) silica, a cyano bounded silica, a phenyl bounded silica, or abiphenyl bounded silica.

In embodiment 1, the sample may be biological sample, a soil sample, ora sample of food stuff. In one particular aspect, the biological sampleis a blood sample, a saliva sample, a lachrymal sample, a urine sample,or a tissue sample. For instance, the blood sample may be a full bloodsample, a plasma sample, or a serum sample.

In a second embodiment, the present invention is directed to aderivatization reagent having a derivatizing agent. In one aspect, thecrown ether derivatizing agent comprises: a crown-ether, a connector,and an analyte-binding functional group. In one particular aspect thecrown-ether comprises a ring, and the ring is a 12-30 membered ring. Ina further aspect, the ring has 12-30 member atoms of which 8-20 atomsare Carbon. In yet a further aspect, the non-carbon member atoms areselected from oxygen, nitrogen, and sulfur. In a particular aspect, thecrown ether is selected from 12 Crown 4, 15 Crown 5, 16 crown 4, 18Crown 6, 21 Crown 7, or 24 Crown 8, optionally having one or moreheteroatoms replacing oxygen.

In one particular aspect, the connector is a C₁-C₁₂ linear, branched,and/or cyclic alkyl group, and even further, the connector may be aphenolic ring fused to the crown ether.

In a particular aspect of embodiment 1, the analyte binding group is anacylating group, 4-Phenyl-1,2,4-triazolin-3,5-dione (PTAD),1,2,4-traizoline-3,5-dione (TAD), an Alkoxylamine, a hydrazide, analcohol, or an amine. For instance, in one particular aspect theacylating group is an acylating agent of Formula 2:

wherein A is the analyte and X is the connector. For instance, in aparticular aspect the analyte binding group is an acylating agent thatis an acyl chloride or acyl halide.

In one particular aspect the crown ether derivatizing agent is selectedfrom the reagents of Group I (laid forth above). In one aspect, thederivatizing agent is MB338. In another aspect, the derivatizing agentis selected from the agents of Group II (laid forth above). In oneaspect, the derivatizing agent MB409. In yet another aspect, thederivatizing agent is selected from the agents of Group III. In still afurther aspect, the derivatizing agent is selected from the agents ofGroup IV.

However, any of the derivatizing agents may be incorporated into aderivatizing reagent and/or used in the methods of the first embodiment.

In particular, where the analyte of embodiment 1 has a primary andsecondary amine, aliphatic hydroxyl, or phenolic hydroxyl group, anacylating agent may be used. In one aspect, the acylating agent is anacyl chloride or acyl halide. In one particular aspect the crown etherderivatizing agent is selected from the reagents of Group I (laid forthabove). In one aspect, the derivatizing agent is MB338. In a furtheraspect, the acylating agent may be used when the analyte is an aminoacid or a metabolite or derivative thereof, an estrogen hormone or ametabolite or derivative thereof, THC or a metabolite or analog thereofor HU210 or a metabolite or derivative thereof.

Further, when the analyte has a ci-diene group, the crown etherderivatizing agent may be selected from the reagents of Group II (laidforth above). In one particular aspect the derivatizing agent is MB409.In one aspect, the analyte may be vitamin D, an analog thereof, or ametabolite thereof as more fully denoted above.

Even further, when the analyte has an aldehyde or ketone the crown etherderivatizing agent may be selected from the agents of Group III (laidforth above). In one particular aspect, the analyte is testosterone or ametabolite thereof, or a ketone or aldehyde containing steroid.

When the analyte is an aliphatic acid, biotin or monomethyl malonic acid(MMA) the crown ether derivatizing agent may be selected from the agentsof Group IV (laid forth above).

In yet a further embodiment, the derivatising agent is incorporated intoa derivatization reagent by the addition of one or more solvents oradditives, which may then be incorporated into a kit for analysis of oneor more particular analytes.

In another embodiment, the mass spectrometry comprises tandem massspectrometry techniques, including LC-MS/MS techniques such asAtmospheric Pressure Chemical Ionization (APCI), Electrospray Ionization(ESI) technique, the use of a triple quadrupole mass spectrometerinstrument in Multiple Reaction Monitoring (MRM), or Selected ReactionMonitoring (SRM), positive-ion mode, a Q1 scan tuned to select aprecursor ion that corresponds to the [M+H]⁺, or [M+NH₄]⁺, or [M+A+H]⁺of the acylated derivatives of the desired analyte for productoptimization, wherein A is a molecular adduct. The detection of theanalyte can be qualitative or quantitative.

Aspects of the present specification can also be described as follows:

-   1. A method for determining the presence of one or more analytes in    a test sample, the method comprising:    -   a) extraction and purification of the analytes from the test        sample with one or more of solid phase extraction, supported        liquid extraction (SLP), and liquid liquid extraction (LLP);    -   b) derivatization of the analytes with a crown-ether        derivatizing agent;    -   c) detection of the derivatized analytes using liquid        chromatography and/or mass spectrometry.-   2. The method of embodiment 1, wherein the analyte is a compound    having a primary amine or a phenolic hydroxyl group.-   3. The method of embodiment 1 or 2, wherein the analyte is a drug, a    hormone, a signaling agent, an amino acid, or a pesticide.-   4. The method of embodiments 1-3, wherein the analyte is a monoamine    neurotransmitter including vitamin D or one of its derivatives or    metabolites, a sex hormone or one of its derivatives or metabolites,    a cannabinoid or one of its derivatives or metabolites, an opiate,    opioid or one of its derivatives or metabolites or an    arylcyclohexylamine or one of its derivatives or metabolites, an    Amphetamine or one of its derivatives or metabolites-   5. The method of embodiment 4, wherein the monoamine    neurotransmitter is Histamine, Tryptamine, Serotonin, or Agmatine.-   6. The method of embodiment 4, wherein the sex hormone or one of its    derivatives or metabolites is an estrogen.-   7. The method of embodiment 4, wherein the derivative of vitamin D    is 25-OH D₃, 25-OH D₂, 24,25-(OH)₂D₃, 1,25-(OH)₂ D₃, and 1,25-(OH)₂    D₂, Cholecalciferol, 25-Hydroxycholecalciferol,    1α,25-Dihydroxycholecalciferol, Ergocalciferol,    1α,25-Dihydroxyergocalciferol, 22,23-Dihydroergocalciferol,    1α,24R,25-Trihydroxycholecalciferol, (6Z)-tacalciol, Tachysterol₃,    Isovitamin D₃, Dihydrotachysterol₃.-   8. The method of embodiment 4, wherein the cannabinoid or one of its    derivatives or metabolites is a Cannabigerol-type (CBG) cannabinoid,    a Cannabichromene-type (CBC) cannabinoid, a Cannabidiol-type (CBD)    cannabinoid, a Cannabinodiol-type (CBND) cannabinoid, a    Tetrahydrocannabinol-type (THC) cannabinoid, a Cannabinol-type (CBN)    cannabinoid, a Cannabitriol-type (CBT) cannabinoid, a    Cannabielsoin-type (CBE) cannabinoid, an Isocannabinoid, a    Cannabicyclol-type (CBL) cannabinoid, a Cannabicitran-type (CBT)    cannabinoid, or a Cannabichromanone-type (CBCN) cannabinoid.-   9. The method of embodiment 4, wherein the opiate, the opioid or the    derivative or metabolite of the opiate or opioid, is morphine,    oripavine, morphinone, hydromorphone, or oxymorphone.-   10. The method of embodiment 4, wherein the opiate, the opioid, or    the derivative or metabolite of the opiate or opioid is a    benzylisoquinoline alkaloid, a semi-synthetic benzylisoquinoline    alkaloid derivative, or an opioid.-   11. The method of embodiment 4, wherein the arylcyclohexylamine or    one of its derivatives or metabolites is Tiletamine, 3-Methoxetamine    (MXE), Methoxyketamine, N-Ethylnorletamine (Ethketamine),    Amphetamine, Ephedrine, or Methamphetamine.-   12. The method of embodiment 4, wherein the Amphetamine or one of    its derivatives or metabolites is Amphetamine (itself),    methamphetamine, ephedrine, cathinone,    3,4-methylenedioxy-N-methylamphetamine (MDMA, “Ecstasy”), and    2,5-Dimethoxy-4-methylamphetamine (DOM, or “STP”).-   13. The method of embodiments 1-12, wherein the solid phase    extraction is performed with an ion exchange column or cartridge.-   14. The method of embodiment 13, wherein the ion exchange column is    a cation exchange column.-   15. The method of embodiment 14, wherein the cation exchange column    is a weak cation exchange column.-   16. The method of embodiment 13, wherein the ion exchange column is    an anion exchange column.-   17. The method of embodiments 1-13, wherein the solid phase    extraction is performed with a reverse phase silica column or    cartridge.-   18. The method of embodiment 17, wherein the reverse phase silica is    an alkyl bounded (C4, C8, C12, or C18) silica, a cyano bounded    silica, a phenyl bounded silica, or a biphenyl bounded silica.-   19. The method of embodiments 1-18, wherein the sample is a    biological sample, a soil sample, or a sample of food stuff.-   20. The method of embodiment 19, wherein the biological sample is a    blood sample, a saliva sample, a lachrymal sample, a urine sample,    or a tissue sample.-   21. The method of embodiment 20, wherein the blood sample is a full    blood sample, a plasma sample, or a serum sample.-   22. The method of embodiments 1-21, wherein the crown-ether    derivatizing agent comprises: a crown-ether, a connector, and an    analyte-binding functional group.-   23. The method of embodiment 22, wherein the crown-ether comprises a    ring, and the ring is a 12-30 membered ring.-   24. The method of embodiment 23, wherein the ring has 12-30 member    atoms of which 8-20 atoms are carbon.-   25. The method of embodiment 24, wherein the non-carbon member atoms    are selected from oxygen, nitrogen, and sulfur.-   26. The method of embodiment 25, wherein the crown ether is selected    from 12 Crown 4, 15 Crown 5, 16 crown 4, 18 Crown 6, 21 Crown 7, or    24 Crown 8, optionally having one or more heteroatoms replacing    oxygen.-   27. The method of embodiments 22-26, wherein the connector is a    C₁-C₁₂ linear, branched, and/or cyclic alkyl group.-   28. The method of embodiments 22-27, wherein the connector is a    phenolic ring fused to the crown ether.-   29. The method of embodiments 22-28, wherein the analyte binding    group is an acylating group, 4-Phenyl-1,2,4-triazolin-3,5-dione    (PTAD), 1,2,4-traizoline-3,5-dione (TAD), an Alkoxylamine, a    hydrazide, an alcohol, or an amine.-   30. The method of any of embodiments 1-29, wherein the acylating    group is an acylating agent of Formula 2:

-    where A is the analyte and X is the connector.-   31. The method of embodiment 30, wherein the acylating agent that is    an acyl chloride or acyl halide.-   32. The method of any of embodiments 1-31, where the crown ether    derivatizing agent is selected from the agents of Group I:

-   33. The method of any of embodiments 1-31, wherein the derivatizing    agent is MB338.-   34. The method of any of embodiments 30-33, wherein the analyte has    a primary and secondary amine, aliphatic hydroxyl, or phenolic    hydroxyl group.-   35. The method of embodiment 34, wherein the analyte is an amino    acid or a metabolite or derivative thereof, an estrogen hormone or a    metabolite or derivative thereof, THC or a metabolite or analog    thereof or HU210 or a metabolite or derivative thereof.-   36. The method of any of embodiments 1-31, wherein the crown ether    derivatizing agent is selected from the agents of Group II:

-   37. The method of any of embodiments 1-31, wherein the derivatizing    agent is MB409.-   38. The method of embodiment 36 or embodiment 37, wherein the    analyte has a ci-diene group.-   39. The method of embodiment 38, wherein the analyte is vitamin D,    an analog thereof, or a metabolite thereof.-   40. The method of any of embodiments 1-31, wherein the derivatizing    agent is selected from the agents of Group III:

-   41. The method of embodiment 40, wherein the analyte has an aldehyde    or ketone.-   42. The method of embodiment 41, wherein the analyte is testosterone    or a metabolite thereof, or a ketone or aldehyde containing steroid.-   43. The method of any of embodiments 1-31, wherein the crown ether    derivatizing agent is selected from the agents of group IV:

-   44. The method of embodiment 43, wherein the analyte is an aliphatic    acid, biotin or monomethyl malonic acid (MMA).-   45. The method of any of embodiments 1-44, wherein the mass    spectrometry comprises tandem mass spectrometry techniques.-   46. The method of any of embodiments 1-45, wherein the mass    spectrometry comprises LC-MS/MS techniques.-   47. The method of embodiment 46, wherein the LC-MS/MS techniques    comprise Atmospheric Pressure Chemical Ionization (APCI), or    Electrospray Ionization (ESI) technique.-   48. The method of embodiment 46, wherein the LC-MS/MS techniques    comprise the use of a triple quadrupole mass spectrometer instrument    in Multiple Reaction Monitoring (MRM), or Selected Reaction    Monitoring (SRM), positive-ion mode.-   49. The method of embodiment 48, wherein the LC-MS/MS techniques    comprise a Q1 scan tuned to select a precursor ion that corresponds    to the [M+H]⁺, or [M+NH₄]⁺, or [M+A+H]⁺ of the acylated derivatives    of the desired analyte for product optimization, wherein A is a    molecular adduct.-   50. The method of any of embodiments 1-49, wherein the detection of    the analyte is qualitative or quantitative.-   51. A derivatization reagent for mass spectrometry having a    derivatizing agent comprising: a crown-ether, a connector, and an    analyte-binding functional group.-   52. The derivatization reagent of embodiment 51, wherein the    crown-ether comprises a ring, and the ring is a 12-30 membered ring.-   53. The derivatization reagent of embodiment 52, wherein the ring    has 12-30 member atoms of which 8-20 atoms are carbon.-   54. The derivatization reagent of embodiment 53, wherein the    non-carbon member atoms are selected from oxygen, nitrogen, and    sulfur.-   55. The derivatization reagent of any of embodiments 51-54, wherein    the crown ether is selected from 12 Crown 4, 15 Crown 5, 16 crown 4,    18 Crown 6, 21 Crown 7, or 24 Crown 8, optionally having one or more    heteroatoms replacing oxygen.-   56. The derivatization reagent of any of embodiments 51-55, wherein    the connector is a C₁-C₁₂ linear, branched, and/or cyclic alkyl    group.-   57. The derivatization reagent of any of embodiments 51-56, wherein    the connector is a phenolic ring fused to the crown ether.-   58. The derivatization reagent of any of embodiments 51-57, wherein    the analyte binding group is an acylating group,    4-Phenyl-1,2,4-triazolin-3,5-dione (PTAD),    1,2,4-traizoline-3,5-dione (TAD), an Alkoxylamine, a hydrazide, an    alcohol, or an amine.-   59. The derivatization reagent of any of embodiments 51-58, wherein    the acylating group is an acylating agent of Formula 2:

-    where A is the analyte and X is the connector.-   60. The derivatization reagent of embodiment 59, wherein the    acylating agent is an acyl chloride or acyl halide.-   61. The derivatization reagent of any of embodiments 51-60, where    the crown ether derivatizing agent is selected from the agents of    Group I:

-   62. The derivatization reagent of any of embodiments 51-61, wherein    the derivatizing agent is MB409.-   63. The derivatization reagent of any of embodiments 51-61, wherein    the crown ether derivatizing agent is selected from the agents of    Group II:

-   64. The derivatization reagent of any of embodiments 51-61, wherein    the derivatizing agent is MB409.-   65. The derivatization reagent of any of embodiments 51-60, wherein    the derivatizing agent is selected from the agents of Group III:

-   66. The derivatization reagent of embodiments 51-58, wherein the    crown ether derivatizing agent is selected from the agents of group    IV:

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofthe disclosed subject matter. These examples should not be construed tolimit any of the embodiments described in the present specification,including those pertaining to the methods for detecting an analyte andkits comprising the components necessary to perform the disclosedmethods.

Example 1 Quantification of 1,25-Dihydroxy-Vitamin D in Plasma SampleCollection

Plasma samples were obtained from human patients' blood. Samples weredrawn (plasma sodium heparin & EDTA) into pre-chilled Vacutainers.Vacutainers were inverted 5× and refrigerated until centrifuged. Plasmawas separated in a refrigerated centrifuge (1000×g for 10 minutes)within 30 minutes of collection and then frozen immediately at −20° C.in plastic vials.

Plasma was thawed and diluted before use in solid phase extraction.

Standards

The blanks, calibration samples, and plasma samples were spiked withinternal standards (e.g., D6-1,25(OH)₂ vitamin D₃ and/or D6-1,25(OH)₂vitamin D₂).

Standard curves were generated with plasma solutions spiked with a knownamount of 1,24-dihydroxy-vitamin D. The spiking solution was seriallydiluted before being added to the plasma taken from the same plasmasample.

Extraction

TRACE-N® (3 cc/15 mg) columns were conditioned with 1.0 ml of methanol,followed by 1 ml deionized water.

Then 200 μL of the Sample was mixed with 1 mL of 28% iso-propyl alcoholin water. 10 μL internal standard was mixed with 1 mL of 28% iso-propylalcohol in water. The Sample/buffer mix was loaded onto the column at apressure of 2-3 psi. The column was washed with 1 ml of 28% iso-propylalcohol in water at 6 psi. The column was dried under a stream ofnitrogen gas for eight minutes at room temperature or heated to 45° C.for 4 min.

The sample was eluted from the column with 0.8 ml of Elution Buffer (20%ethyl acetate in hexane).

Solvent was evaporated under a stream of nitrogen gas with heating to40° C. for 4 min. The extract was reconstituted with ethylacetate (100μL).

The extract was derivatized as follows:

MB409 (the crown-ether derivatization agent) in ethylacetate (1 mg/mL, 5μL) was added to the reconstituted extract and vortexed for 20 seconds.The derivatization reaction was permitted to proceed for 30 minutes atroom temperature. The reaction is stopped with the addition of 20 μLmethanol and vortexing for 10 seconds. The derivatized analyte wasconcentrated by streaming with nitrogen gas for 3 minutes. Theconcentrated analyte is reconstituted with 50 μL acetonitrile/NH₄CO₂H(50 mM) (at 3:1).

The reconstituted analyte was loaded onto an autosampler for LCMSanalysis.

LC-MS/MS Analysis

10 μL of the solution obtained from the ammonium formate reaction wasautomatically injected into a TARGA® C18 3 μm particle size 50×2.1 mmanalytical column. A binary HPLC gradient was applied to the analyticalcolumn to separate the crown ether derivatives of 1,25-dihydroxy vitaminD from other analytes contained in the sample. Mobile phase A was 5.0 mMammonium formate with 0.1% formic acid pH 3.0 and mobile phase B wasAcetonitrile with 0.1% formic acid. The HPLC gradient proceeded at atemperature of 35° C. with a flow rate of 600 μl/min over five minutesas follows:

Gradient: Time (min) B (%) 0.01 50 2.00 50 2.05 98 4.00 98 4.50 50 5.0050

MS/MS was performed using an AB Sciex 4000 QTrap for detection coupledto an Agilent 1200 binary LC pump controlled by Analyst Software Version1.52 (ABI-SCIEX, Toronto, Canada). Analyte exiting the HPLC analyticalcolumn through the mobile phase flowed to the heated nebulizer interfaceof the MS/MS analyzer. The solvent/analyte mixture was converted tovapor in the heated tubing of the interface. Analytes in the nebulizedsolvent were ionized by heated Electrospray Ionization source.

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of parent ions generated from one of the analytes.Ions entering quadrupole 2 (Q2) collided with collision gas to generateion fragments, which were passed to quadrupole 3 (Q3) for furtherselection. Simultaneously, the same process using isotope dilution massspectrometry was carried out with internal standards, D6-1,25(OH)₂vitamin D₃ and/or D6-1,25(OH)₂ vitamin D₂. The following masstransitions were used for detection and quantitation of1,25-dihydroxy-vitamin D (and its corresponding internal standards)during validation on positive polarity from the same sample injection.

Results:

The results demonstrate an automated method for extracting1,25-dihydroxy vitamin D. The method gives good linear response to bothcompounds from 5 pg mL-20 ng/mL of the standard curve, with an LOD of 10pg/mL of plasma for 1,25-dihydroxy vitamin D by using an AB Sciex 4000QTrap for detection coupled to an Agilent 1200 binary LC pump.

Example 2 Quantification of THC or HU210 in Oral Fluid Sample Collection

Oral samples were taken from humans using the QUANTISAL™ (Immunalysis,California). Saliva was taken from patients using the collection devicefrom QUANTISAL, and specimens were refrigerated and ultimately frozen.Saliva was thawed before use in the present quantification methods.

Standards

The blanks, calibration samples, and oral samples were spiked withinternal standards (e.g., THC-d3).

Standard curves were generated with saliva solutions spiked with a knownamount of THC or HU210 (a synthetic cannabinoid). The spiking solutionwas serially diluted before being added to the saliva taken from thesame oral sample.

Extraction

TRACE-N® (3 cc/15 mg) columns were conditioned with 0.5 ml of methanol,followed by 0.5 ml 0.1% HCl (6M) in water.

Then 300 uL of the oral fluid in QUANTISAL™ buffer (25% v/v). 10 μLinternal standard was added and 600 uL of 100 mM pH6 phosphate buffer.The Sample/buffer mix was loaded onto the column at a pressure of 2-3psi. The column was washed with 1 mL of DI water, then 1 ml of 20%methanol in water at 2-3 psi. The column was dried under a stream ofnitrogen gas for eight minutes at room temperature or heated to 45° C.for 4 min.

The sample was eluted from the column with 0.8 ml of Elution solvent(20% ethyl acetate in hexane).

Solvent was evaporated under a stream of nitrogen gas with heating to40° C. for 8 min. The extract was reconstituted with acetonitrile (50μL) K₂CO₃ (50 mM, 25 μL). MB388 (the crown-ether derivatization agent)in acetonitrile (10 mg/mL, 5 μL) was added to the reconstituted extractand vortexed for 20 seconds. The derivatization reaction was permittedto proceed for 30 minutes at room temperature. The reaction is stoppedwith the addition of 20 μL ammonium bicarbonate (300 mM).

The reconstituted analyte was loaded onto an autosampler for LCMSanalysis.

LC-MS/MS Analysis

10 μL of the solution obtained from the ammonium formate reaction wasautomatically injected into a RAPTOR® C18 (2.7 μm particle size 50×2.1mm analytical column. A binary HPLC gradient was applied to theanalytical column to separate the crown ether derivatives of THC orHU210 from other analytes contained in the sample. Mobile phase A was5.0 mM ammonium formate with 0.1% formic acid pH 3.0 and mobile phase Bwas Acetonitrile with 0.1% formic acid. The HPLC gradient proceeded at atemperature of 35° C. with a flow rate of 600 μl/min over five minutesas follows:

Gradient: Time (min) B (%) 0.01 50 2.00 50 2.05 98 4.00 98 4.50 50 5.0050

MS/MS was performed using an API 5000 triple quardrupole massspectrometer controlled by Analyst Software Version 1.52 (ABI-SCIEX,Toronto, Canada). Analyte exiting the HPLC analytical column through themobile phase flowed to the heated nebulizer interface of the MS/MSanalyzer. The solvent/analyte mixture was converted to vapor in theheated tubing of the interface. Analytes in the nebulized solvent wereionized by heated Electrospray Ionization source.

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of parent ions generated from one of the analytes.Ions entering quadrupole 2 (Q2) collided with collision gas to generateion fragments, which were passed to quadrupole 3 (Q3) for furtherselection. Simultaneously, the same process using isotope dilution massspectrometry was carried out with internal standards, (−)-□-9-THC (D3).The following mass transitions were used for detection and quantitationof THC or HU210 (and the same internal standards) during validation onpositive polarity from the same sample injection.

TABLE 3 MRM Transitions for THC Compound Polarity Precursor m/z Productm/z THC MB338 Positive 670 339 THC MB338 Positive 670 163 THC MB338Positive 670 107 THC d3 MB338 Positive 673 339 THC d3 MB338 Positive 673163 THC d3 MB338 Positive 673 107 HU210 MB338 Positive 742 337 HU210MB338 Positive 742 339 HU210 MB338 Positive 742 163

Results:

Table 4 shows the results of the standard curve of THC.

TABLE 4 THC Standard Curve Data THC Standard Curve Low 087 Low ContStandard Mar. 26, 2015 8.3 87 2.32e+006 2.47e+006 100 9.39e−001 95.9 088Low Cont Standard Mar. 26, 2015 8.3 88 2.62e+006 2.61e+006 100 1.00e+000103 089 Low Cont Standard Mar. 26, 2015 8.4 89 2.71e+006 2.78e+006 1009.74e−001 99.5 090 Low Cont Standard Mar. 26, 2015 8.4 90 2.81e+0062.79e+006 100 1.01e+000 103 091 Low Cont Standard Mar. 26, 2015 8.5 912.93e+006 3.01e+006 100 9.71e−001 99.1 032 Low Cont Unknown Mar. 26,2015 3.1 32 2.44e+006 3.15e+006 N/A 7.73e−001 79 033 Low Cont UnknownMar. 26, 2015 3.1 33 2.54e+006 3.17e+006 N/A 8.03e−001 82 034 Low ContUnknown Mar. 26, 2015 3.2 34 2.19e+006 2.72e+006 N/A 8.02e−001 81.9 035Low Cont Unknown Mar. 26, 2015 3.2 35 2.04e+006 2.48e+006 N/A 8.23e−00184 036 Low Cont Unknown Mar. 26, 2015 3.3 36 2.74e+006 3.33e+006 N/A8.22e−001 83.9 Average % Recovery 82.16 THC Standard Curve HighCalculated Vial Analyte IS Peak Analyte Area Concentration Sample Nam

Sample Type Acquisition D Position Peak Area Conc Ratio (ng/mL) 092 HighCon Standard Mar. 26, 2015 8.5 92 1.94e+007 2.84e+006 100 6.81e+000 104093 High Con Standard Mar. 26, 2015 9.0 93 2.21e+007 3.37e+006 1006.57e+000 99.9 094 High Con Standard Mar. 26, 2015 9.1 94 2.00e+0073.19e+006 100 6.29e+000 95.7 095 High Con Standard Mar. 26, 2015 9.1 952.09e+007 3.14e+006 100 6.66e+000 101 096 High Con Standard Mar. 26,2015 9.2 96 1.91e+007 2.93e+006 100 6.54e+000 99.5 045 High Con UnknownMar. 26, 2015 4.2 45 1.63e+007 2.81e+006 N/A 5.81e+000 88.3 046 High ConUnknown Mar. 26, 2015 4.3 46 1.48e+007 2.43e+006 N/A 6.11e+000 92.9 047High Con Unknown Mar. 26, 2015 4.3 47 1.59e+007 2.55e+006 N/A 6.25e+00095 048 High Con Unknown Mar. 26, 2015 4.4 48 1.68e+007 2.80e+006 N/A6.00e+000 91.2 Average % Recovery 91.85

indicates data missing or illegible when filedTable 5 shows the standard curve data for H210.

TABLE 5 H210 Data HU210 Curve Low 087 Low Cont Standard Mar. 26, 20158.3 87 7.73e+005 2.47e+006 100 3.12e−001 103 088 Low Cont Standard Mar.26, 2015 8.3 88 8.37e+005 2.61e+006 100 3.21e−001 105 089 Low ContStandard Mar. 26, 2015 8.4 89 8.51e+005 2.78e+006 100 3.06e−001 101 090Low Cont Standard Mar. 26, 2015 8.4 90 8.29e+005 2.79e+006 100 2.97e−00197.6 091 Low Cont Standard Mar. 26, 2015 8.5 91 8.62e+005 3.01e+006 1002.86e−001 93.8 032 Low Cont Unknown Mar. 26, 2015 3.1 32 8.53e+0053.15e+006 N/A 2.70e−001 88.8 033 Low Cont Unknown Mar. 26, 2015 3.1 337.91e+005 3.17e+006 N/A 2.50e−001 82 034 Low Cont Unknown Mar. 26, 20153.2 34 6.57e+005 2.72e+006 N/A 2.41e−001 79.1 035 Low Cont Unknown Mar.26, 2015 3.2 35 5.78e+005 2.48e+006 N/A 2.33e−001 76.5 036 Low ContUnknown Mar. 26, 2015 3.3 36 7.16e+005 3.33e+006 N/A 2.15e−001 70.6Average % Recovery 79.4 Hu210 Curve High Calculated Vial Analyte IS PeakAnalyte Area Concentration Sample Nam

Sample Type Acquisition D Position Peak Area Conc Ratio (ng/mL) 092 HighCon Standard Mar. 26, 2015 8.5 92 6.08e+006 2.84e+006 100 2.14e+000 103093 High Con Standard Mar. 26, 2015 9.0 93 6.89e+006 3.37e+006 1002.04e+000 98 094 High Con Standard Mar. 26, 2015 9.1 94 6.37e+0063.19e+006 100 2.00e+000 95.9 095 High Con Standard Mar. 26, 2015 9.1 956.43e+006 3.14e+006 100 2.05e+000 98.2 096 High Con Standard Mar. 26,2015 9.2 96 6.43e+006 2.93e+006 100 2.20e+000 105 045 High Con UnknownMar. 26, 2015 4.2 45 5.63e+006 2.81e+006 N/A 2.01e+000 96.3 046 High ConUnknown Mar. 26, 2015 4.3 46 5.05e+006 2.43e+006 N/A 2.08e+000 99.7 047High Con Unknown Mar. 26, 2015 4.3 47 4.61e+006 2.55e+006 N/A 1.81e+00086.8 048 High Con Unknown Mar. 26, 2015 4.4 48 5.11e+006 2.80e+006 N/A1.82e+000 87.4 Average % Recovery 92.55

indicates data missing or illegible when filedTables 4 and 5 show the data representing the percent recovery of THC orHU210 from a plasma sample spiked with 250 fg/mL-2 ng/mL THC or HU210.LLOD: 200 fg on column, LLOQ: 400 fg on column. The assay LLOD was 20pg/mL and LLOQ was 40 pg/mL, with extraction of 75 uL of saliva.Recovery of the analyte was 80-92%. FIGS. 2A and 2B show the linearregression for THC and HU210.

The results describe an automated method for extracting THC and HU210from oral fluid. The method gives good linear response to both compoundsfrom 250 fg mL-2 ng/mL of the standard curve, with an LOD of 1 pg/mL oforal fluid for both THC and HU210 by using an AB Sciex 5000 fordetection coupled to a pair of Shimadzu 20AD LC pumps.

Example 3 Quantification ofEstradiol/Estriol/Estrone/17β-Estradiol-2,3,4-¹³C₃ in Plasma SampleCollection

Plasma samples were obtained from human patients' blood. Samples weredrawn (plasma sodium heparin & EDTA) into pre-chilled Vacutainers.Vacutainers were inverted 5× and refrigerated until centrifuged. Plasmawas separated in a refrigerated centrifuge (1000×g for 10 minutes)within 30 minutes of collection and then frozen immediately at −20° C.in plastic vials.

Plasma was thawed and diluted before use in solid phase extraction.

Standards

The blanks, calibration samples, and plasma samples were spiked withinternal standards (e.g., 17β-estradiol-2,3,4-¹³C₃).

Standard curves were generated with plasma solutions spiked with a knownamount of estradiol or estrone. The spiking solution was seriallydiluted before being added to the plasma taken from the same plasmasample.

Extraction

MAESTRO® A (1 cc/15 mg) columns were conditioned with 1.0 mL ofmethanol, followed by 0.5 mL deionized water.

Then 100 μL of the Sample was mixed with 0.5 mL of water. Another 0.5 mLof water was added. The Sample/buffer mix was loaded onto the column ata pressure of 2-3 psi. The column was washed with 1 ml of 20% iso-propylalcohol in water at 6 psi. The column was dried under a stream ofnitrogen gas for 10 minutes at room temperature or heated to 45° C. for5 min.

The sample was eluted from the column with 0.7 ml of Elution solvent(80:20 hexane:ethyl acetate).

Solvent was evaporated under a stream of nitrogen gas with heating to40° C. for 4 min. The extract was reconstituted with acetonitrile (50μL) K₂CO₃ (50 mM, 25 μL).

The extract was derivatized as follows:

MB388 (the crown-ether derivatization agent) in acetonitrile (10 mg/mL,5 μL) was added to the reconstituted extract and vortexed for 20seconds. The derivatization reaction was permitted to proceed for 30minutes at room temperature. The reaction is stopped with the additionof 20 μL ammonium bicarbonate (300 mM).

The reconstituted analyte was loaded onto an autosampler for LCMSanalysis.

LC-MS/MS Analysis

10 μL of the solution obtained from the ammonium formate reaction wasautomatically injected into a Targa® C18 (3 μm particle size 50×2.1 mmanalytical column. A binary HPLC gradient was applied to the analyticalcolumn to separate the crown ether derivatives of Estradiol or Estronefrom other analytes contained in the sample. Mobile phase A was 5.0 mMammonium formate with 0.1% formic acid pH 3.0 and mobile phase B wasAcetonitrile with 0.1% formic acid. The HPLC gradient proceeded at atemperature of 35° C. with a flow rate of 600 μl/min over five minutesas follows:

Gradient: Time (min) B (%) 0.01 50 3.00 100 4.00 100 4.5 50 5 50

MS/MS was performed using an API 4000 QTRAP triple quardrupole massspectrometer coupled to an Agilent 1200 binary HPLC pump, controlled byAnalyst Software Version 1.52 (ABI-SCIEX, Toronto, Canada). Analyteexiting the HPLC analytical column through the mobile phase flowed tothe heated nebulizer interface of the MS/MS analyzer. Thesolvent/analyte mixture was converted to vapor in the heated tubing ofthe interface. Analytes in the nebulized solvent were ionized by heatedElectrospray Ionization source.

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of parent ions generated from one of the analytes.Ions entering quadrupole 2 (Q2) collided with collision gas to generateion fragments, which were passed to quadrupole 3 (Q3) for furtherselection. Simultaneously, the same process using isotope dilution massspectrometry was carried out with internal standards.

The following mass transitions were used for detection and quantitationof Estradiol or Estrone (and their corresponding internal standards)during validation on positive polarity from the same sample injection.

TABLE 6 Mass Transitions Compound Polarity Precursor m/z Product m/zEstrone MB338 Positive 626.46 338.8 Estrone MB338 Positive 626.46 163Estradiol MB338 Positive 628.56 339.1 Estradiol MB338 Positive 628.56162.9 Estriol MB338 Positive 644.28 339 Estriol MB338 Positive 644.28162.8 17β-Estradiol-2,3,4-13C3 Positive 631.399 339 MB33817β-Estradiol-2,3,4-13C3 Positive 631.399 163 MB33817β-Estradiol-2,3,4-13C3 Positive 631.399 107 MB338

TABLE 7 Estrone and Estradiol Mass and Dwell Time Parent Mass FragmentDwell Time Compound (m/z) (m/z) (ms) DP EP CP CXP Estrone MB338 1 626.46338.8 100 86 10 33 8 Estrone MB338 2 626.46 163 100 86 10 59 12Estradiol MB338 1 628.56 339.1 100 86 10 33 10 Estradiol MB338 2 628.56162.9 100 86 10 59 12 17β-Estradiol-2,3,4- 631.399 339 100 96 10 33 813C3 MB338 1 17β-Estradiol-2,3,4- 631.399 163 100 96 10 63 26 13C3 MB3381

TABLE 8 Recovery Data Analyte Peak Sample Analyte Area (counts) %Recovery Std Curve 200 pg/mL Estrone 32000 201 626.46 > 338.8 Std Curve200 pg/mL Estradiol 30900 184 628.56 > 339.1 Patient Sample 1 Estrone7510 48.1 626.46 > 338.8 Patient Sample 1 Estradiol 2560 20.6 628.56 >339.1 Patient Sample 4 Estrone 18100 112 626.46 > 338.8 Patient Sample 4Estradiol 20100 154 628.56 > 339.1 Patient Sample 6 Estrone 702 6.63626.46 > 338.8 Patient Sample 6 Estradiol 1610 15.2 628.56 > 339.1

The method is an automated method for extracting Estrone (E1) and17β-estradiol (E2) from 100 uL plasma. The method gives good linearresponse to both compounds from 5-500 pg/mL, with an LOD of 5 pg/mL forEstradiol and 10 ng for Estrone using an AB Sciex Qtrap 4000 fordetection coupled to an Agilent 1200 HPLC (see FIGS. 3A and 3B).

Further tests have demonstrated detection of estrone and estradiol inthe pictogram range with a LOQ of 10 pg/mL for both compounds. Absoluterecovery for Estrone and Estradiol were >90% (see FIG. 4A for additionalchromatograms).

To test for variability ten replicate samples were spiked to aconcentration of 100 pg/mL. Subsequent extraction and analysis showed a7.6% and 8.6% CSV for estrone and estradiol (data not shown).

Finally, plasma from ten volunteers was measured in duplicate andanalyzed for estradiol and estrone. The level of estrone and estradiolin all samples corresponded to physiological levels found in healthyadults (See FIG. 4B). Females (such as patient 4) have a wide range ofestrogen levels depending on fertility and health factors, and the assayis able to measure the full extent of these values.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particular compound,composition, article, apparatus, methodology, protocol, and/or reagent,etc., described herein, unless expressly stated as such. In addition,those of ordinary skill in the art will recognize that certain changes,modifications, permutations, alterations, additions, subtractions andsub-combinations thereof can be made in accordance with the teachingsherein without departing from the spirit of the present specification.It is therefore intended that the following appended claims and claimshereafter introduced are interpreted to include all such changes,modifications, permutations, alterations, additions, subtractions andsub-combinations as are within their true spirit and scope.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. For instance, as massspectrometry instruments can vary slightly in determining the mass of agiven analyte, the term “about” in the context of the mass of an ion orthe mass/charge ratio of an ion refers to +/−0.50 atomic mass unit. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalindication should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Use of the terms “may” or “can” in reference to an embodiment or aspectof an embodiment also carries with it the alternative meaning of “maynot” or “cannot.” As such, if the present specification discloses thatan embodiment or an aspect of an embodiment may be or can be included aspart of the inventive subject matter, then the negative limitation orexclusionary proviso is also explicitly meant, meaning that anembodiment or an aspect of an embodiment may not be or cannot beincluded as part of the inventive subject matter. In a similar manner,use of the term “optionally” in reference to an embodiment or aspect ofan embodiment means that such embodiment or aspect of the embodiment maybe included as part of the inventive subject matter or may not beincluded as part of the inventive subject matter. Whether such anegative limitation or exclusionary proviso applies will be based onwhether the negative limitation or exclusionary proviso is recited inthe claimed subject matter.

Notwithstanding that the numerical ranges and values setting forth thebroad scope of the invention are approximations, the numerical rangesand values set forth in the specific examples are reported as preciselyas possible. Any numerical range or value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements. Recitation of numerical rangesof values herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar references used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Further, ordinal indicators—such as “first,” “second,” “third,”etc.—for identified elements are used to distinguish between theelements, and do not indicate or imply a required or limited number ofsuch elements, and do not indicate a particular position or order ofsuch elements unless otherwise specifically stated. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the presentinvention and does not pose a limitation on the scope of the inventionotherwise claimed. No language in the present specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

When used in the claims, whether as filed or added per amendment, theopen-ended transitional term “comprising” (and equivalent open-endedtransitional phrases thereof like including, containing and having)encompasses all the expressly recited elements, limitations, stepsand/or features alone or in combination with unrecited subject matter;the named elements, limitations and/or features are essential, but otherunnamed elements, limitations and/or features may be added and stillform a construct within the scope of the claim. Specific embodimentsdisclosed herein may be further limited in the claims using theclosed-ended transitional phrases “consisting of” or “consistingessentially of” in lieu of or as an amended for “comprising.” When usedin the claims, whether as filed or added per amendment, the closed-endedtransitional phrase “consisting of” excludes any element, limitation,step, or feature not expressly recited in the claims. The closed-endedtransitional phrase “consisting essentially of” limits the scope of aclaim to the expressly recited elements, limitations, steps and/orfeatures and any other elements, limitations, steps and/or features thatdo not materially affect the basic and novel characteristic(s) of theclaimed subject matter. Thus, the meaning of the open-ended transitionalphrase “comprising” is being defined as encompassing all thespecifically recited elements, limitations, steps and/or features aswell as any optional, additional unspecified ones. The meaning of theclosed-ended transitional phrase “consisting of” is being defined asonly including those elements, limitations, steps and/or featuresspecifically recited in the claim whereas the meaning of theclosed-ended transitional phrase “consisting essentially of” is beingdefined as only including those elements, limitations, steps and/orfeatures specifically recited in the claim and those elements,limitations, steps and/or features that do not materially affect thebasic and novel characteristic(s) of the claimed subject matter.Therefore, the open-ended transitional phrase “comprising” (andequivalent open-ended transitional phrases thereof) includes within itsmeaning, as a limiting case, claimed subject matter specified by theclosed-ended transitional phrases “consisting of” or “consistingessentially of.” As such embodiments described herein or so claimed withthe phrase “comprising” are expressly or inherently unambiguouslydescribed, enabled and supported herein for the phrases “consistingessentially of” and “consisting of.”

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

1. A method for determining the presence of one or more analytes in atest sample, the method comprising: a) extraction and purification ofthe one or more analytes from the test sample with one or more of solidphase extraction, supported liquid extraction (SLP), and liquid liquidextraction (LLP); b) derivatization of the one or more analytes with acrown-ether derivatizing agent; c) detection of the one or morederivatized analytes using liquid chromatography and/or massspectrometry.
 2. The method of claim 1, wherein the one or more analytesis a drug, a hormone, a signaling agent, an amino acid, or a pesticide.3. The method of claim 1, wherein the one or more analytes is amonoamine neurotransmitter including vitamin D or one of its derivativesor metabolites, a sex hormone or one of its derivatives or metabolites,a cannabinoid or one of its derivatives or metabolites, an opiate,opioid or one of its derivatives or metabolites or anarylcyclohexylamine or one of its derivatives or metabolites, anAmphetamine or one of its derivatives or metabolites
 4. The method ofclaim 3, wherein the monoamine neurotransmitter is Histamine,Tryptamine, Serotonin, or Agmatine; wherein the sex hormone or one ofits derivatives or metabolites is an estrogen; wherein the derivative ofvitamin D is 25-OH D₃, 25-OH D₂, 24,25-(OH)₂ D₃, 1,25-(OH)₂ D₃, and1,25-(OH)₂ D₂, Cholecalciferol, 25-Hydroxycholecalciferol,1α,25-Dihydroxycholecalciferol, Ergocalciferol,1α,25-Dihydroxyergocalciferol, 22,23-Dihydroergocalciferol,1α,24R,25-Trihydroxycholecalciferol, (6Z)-tacalciol, Tachysterol₃,Isovitamin D₃, Dihydrotachysterol₃; wherein the cannabinoid or one ofits derivatives or metabolites is a Cannabigerol-type (CBG) cannabinoid,a Cannabichromene-type (CBC) cannabinoid, a Cannabidiol-type (CBD)cannabinoid, a Cannabinodiol-type (CBND) cannabinoid, aTetrahydrocannabinol-type (THC) cannabinoid, a Cannabinol-type (CBN)cannabinoid, a Cannabitriol-type (CBT) cannabinoid, a Cannabielsoin-type(CBE) cannabinoid, an Isocannabinoid, a Cannabicyclol-type (CBL)cannabinoid, a Cannabicitran-type (CBT) cannabinoid, or aCannabichromanone-type (CBCN) cannabinoid; wherein the opiate, theopioid or the derivative or metabolite of the opiate or opioid, ismorphine, oripavine, morphinone, hydromorphone, oxymorphone, abenzylisoquinoline alkaloid, a semi-synthetic or a benzylisoquinolinealkaloid derivative; wherein the arylcyclohexylamine or one of itsderivatives or metabolites is Tiletamine, 3-Methoxetamine (MXE),Methoxyketamine, N-Ethylnorletamine (Ethketamine), Amphetamine,Ephedrine, or Methamphetamine; or wherein the Amphetamine or one of itsderivatives or metabolites is Amphetamine (itself), methamphetamine,ephedrine, cathinone, 3,4-methylenedioxy-N-methylamphetamine (MDMA,“Ecstasy”), and 2,5-Dimethoxy-4-methylamphetamine (DOM, or “STP”). 5.The method of claim 1, wherein the solid phase extraction is performedwith an ion exchange column or cartridge, wherein the ion exchangecolumn is a cation exchange column, a weak cation exchange column or ananion exchange column.
 6. The method of claim 1, wherein the solid phaseextraction is performed with a reverse phase silica column or cartridge.7. The method of claim 6, wherein the reverse phase silica is an alkylbounded (C4, C8, C12, or C18) silica, a cyano bounded silica, a phenylbounded silica, or a biphenyl bounded silica.
 8. The method of claim 1,wherein the sample is a biological sample, a soil sample, or a sample offood stuff.
 9. The method of claim 8, wherein the biological sample is ablood sample, a saliva sample, a lachrymal sample, a urine sample, or atissue sample.
 10. The method of claim 9, wherein the blood sample is afull blood sample, a plasma sample, or a serum sample.
 11. The method ofclaim 1, wherein the crown-ether derivatizing agent comprises: acrown-ether, a connector, and an analyte-binding functional group. 12.The method of claim 11, wherein the crown-ether comprises a ring, andthe ring is a 12-30 membered ring.
 13. The method of claim 12, whereinthe ring has 12-30 member atoms of which 8-20 atoms are carbon andwherein the non-carbon member atoms are selected from oxygen, nitrogen,and sulfur.
 14. The method of claim 11, wherein the crown ether isselected from 12 Crown 4, 15 Crown 5, 16 crown 4, 18 Crown 6, 21 Crown7, or 24 Crown 8, optionally having one or more heteroatoms replacingoxygen.
 15. The method of claim 11, wherein the connector is a C₁-C₁₂linear, branched, and/or cyclic alkyl group or a phenolic ring fused tothe crown ether.
 16. The method of claim 11, wherein the analyte bindinggroup is an acylating group, 4-Phenyl-1,2,4-triazolin-3,5-dione (PTAD),1,2,4-traizoline-3,5-dione (TAD), an Alkoxylamine, a hydrazide, analcohol, or an amine.
 17. The method of claim 16, wherein the acylatinggroup is an acylating agent of Formula 2:

where A is the analyte and X is the connector.
 18. The method of claim17, wherein the acylating agent that is an acyl chloride or acyl halide.19. The method of claim 1, where the crown ether derivatizing agent isselected from the agents of Group I, II, III, or IV:


20. The method of claim 19, wherein the derivatizing agent is MB338 orMB409.
 21. A derivatization reagent for mass spectrometry having aderivatizing agent comprising: a crown-ether, a connector, and ananalyte-binding functional group.